Methods of obtaining tumor-specific t cell receptors

ABSTRACT

Provided methods of obtaining a plurality of T cell receptors specifically recognizing a target tumor antigen peptide from an individual that has clinically benefitted from an immunotherapy, such as Multiple Antigen Specific Cell Therapy. Also provided tumor-specific TCRs, engineered immune cells expressing the TCRs and methods of treating a disease using the engineered immune cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase filing under 35 U.S.C. § 371 ofInternational Application No. PCT/US2019/082408, filed internationallyon Apr. 12, 2019, which claims the priority benefit of InternationalPatent Application No. PCT/CN2018/082947, filed Apr. 13, 2018, thecontents of which are incorporated herein by reference in theirentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 776902000300SEQLIST.TXT,date recorded: Oct. 9, 2020, size: 432 KB).

FIELD OF THE INVENTION

The present invention relates to the field of cancer immunotherapy. Inparticular, this invention provides methods of obtaining TCRs,tumor-specific TCRs, engineered immune cells, pharmaceuticalcompositions and kits for treating cancer.

BACKGROUND OF THE INVENTION

Unlike hematological cancer, solid tumors lack cell surface antigens tobe recognized by typical Chimeric Antigen Receptors (CARs).Intracellular antigens from solid tumors must be presented by HLAmolecules as HLA-antigen epitope complexes on the cell surface, whichare specifically recognized by T cell receptors (TCRs) on the surface oftumor-specific T cells, thereby eliciting anti-tumor cytotoxicity by thetumor-specific T cells.

By February 2018, there are 75 clinical trials on adoptive immune cellsengineered with tumor-specific TCRs for treating cancer. Targets includetumor-associated antigens, tumor-associated viral antigens andneoantigens. Indicates include many common types of solid tumors.Examples of TCR-T cells currently in clinical trials include TCR-Tstargeting WT1/HLA-A*0201 complex (Juno Therapeutics) for treating acutemyeloid leukemia (AML) and non-small cell lung cancer; TCR-Ts targetingMAGE-A3/A6/HLA-DPB1 *0401 complex (Kite) for treating malignant solidtumors; and TCR-Ts targeting MAGE-A4/HLA-A*0201(Adaptimmune) fortreating malignant solid tumors.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods, compositions and kits forobtaining one or more T cell receptors (TCRs) that specificallyrecognize a tumor antigen peptide from an individual that has clinicallybenefitted from an immunotherapy such as Multiple Antigen Specific CellTherapy (“MASCT”).

One aspect of the present application provides a method of obtaining aplurality of T cell receptors (TCRs) specifically recognizing a targettumor antigen peptide, comprising: a) a first co-culturing stepcomprising co-culturing a first population of dendritic cells (DCs)loaded with the target tumor antigen peptide with a population of Tcells from an individual to obtain a first co-culture; b) an enrichmentstep comprising subjecting the first co-culture to an enrichment processto obtain enriched activated T cells; c) a second co-culturing stepcomprising co-culturing the enriched activated T cells with a secondpopulation of DCs loaded with the target tumor antigen peptide to obtaina population of tumor antigen-specific T cells, wherein at least about10% of the tumor antigen-specific T cells specifically responds to thetarget tumor antigen peptide; and d) a sequencing step, comprisingsubjecting the tumor antigen-specific T cells to next-generationsequencing to identify a plurality of pairs of genes encoding TCRα andTCRβ, thereby providing the plurality of T cell receptors based onpaired genes encoding TCRα and TCRβ; wherein the individual hasclinically benefitted from a Multiple Antigen Specific Cell Therapy(MASCT) comprising administering to the individual an effective amountof activated T cells prepared by co-culturing a population of T cellswith a population of dendritic cells loaded with a plurality of tumorantigen peptides comprising the target tumor antigen peptide. In someembodiments, the first co-culturing step is carried out for about 1 toabout 3 days prior to the enrichment step. In some embodiments, theratio between the population of T cells to the first population of DCsloaded with the target tumor antigen peptide is no more than about 30:1(such as about 10:1 to about 20:1, or about 15:1 or about 20:1). In someembodiments, the population of T cells in the first co-culturing step ispresent in PBMCs. In some embodiments, the first population of DCsloaded with the target tumor antigen peptide and the population of Tcells are co-cultured in a first co-culture medium comprising one ormore cytokines (e.g., IL-2 or a plurality of cytokines) and an immunecheckpoint inhibitor. In some embodiments, the first co-culture mediumcomprises IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1 antibody. In someembodiments, the first co-culture medium comprises IL-2 and an anti-PD-1antibody.

In some embodiments according to any one of the methods described above,the enrichment step comprises contacting the first co-culture withantigen presenting cells (APCs) loaded with the target tumor antigenpeptide to obtain a stimulated co-culture, and isolating from thestimulated co-culture an enriched population of activated T cells usinga ligand that specifically recognizes a cytokine. In some embodiments,the cytokine is IFNy. In some embodiments, the ratio between theenriched population of activated T cells and the second population ofDCs loaded with the target tumor antigen peptide is about 1:1 to about20:1. In some embodiments, the enriched population of activated T cellsand the second population of DCs loaded with the target tumor antigenpeptide are co-cultured for about 12 to 25 days.

In some embodiments according to any one of the methods described above,the second co-culturing step comprises co-culturing the secondpopulation of DCs loaded with the target tumor antigen peptide with theenriched population of activated T cells in an initial second co-culturemedium comprising an immune checkpoint inhibitor and optionally one ormore cytokines (e.g., IL-2 or a plurality of cytokines) to provide asecond co-culture; and adding an anti-CD3 antibody (e.g., OKT3) andoptionally one or more cytokines (e.g., IL-2 or a plurality ofcytokines) to the second co-culture to obtain a population of tumorantigen-specific T cells. In some embodiments, the anti-CD3 antibody isadded to the second co-culture no more than about 3 days after thesecond co-culturing step starts. In some embodiments, the anti-CD3antibody is OKT3. In some embodiments, the one or more cytokines isadded to the second co-culture no more than about 3 days (e.g., about 2days) after the second co-culturing step starts. In some embodiments,the one or cytokines comprises IL-2. In some embodiments, the immunecheckpoint inhibitor is an anti-PD-1 antibody (e.g., SHR-1210). In someembodiments, the initial second co-culture medium comprises IL-2, IL-7,IL-15 and IL-21 and an anti-PD-1 antibody.

In some embodiments according to any one of the methods described above,the method further comprises a third co-culturing step comprisingco-culturing a population of the tumor antigen-specific T cells with apopulation of antigen presenting cells (APCs) loaded with target tumorantigen peptide to obtain a second population of tumor antigen-specificT cells, wherein the second population of tumor antigen-specific T cellsare subjected to next-generation sequencing in the sequencing step. Insome embodiments, the APCs are PBMCs, DCs, or cell line APCs. In someembodiments, the ratio between the population of tumor antigen-specificT cells and the population of APCs loaded with the target tumor antigenpeptide is about 1:1 to about 20:1. In some embodiments, the populationof tumor antigen-specific T cells and the population of APCs loaded withthe target tumor antigen peptide are co-cultured for about 5 to 9 days.In some embodiments, the population of tumor antigen-specific T cellsand the population of APCs loaded with the target tumor antigen peptideare co-cultured in a third co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines) and an anti-CD3antibody. In some embodiments, the third co-culture medium comprisesIL-2, IL-7, IL-15 and OKT3. In some embodiments, the third co-culturemedium comprises IL-2 and OKT3. In some embodiments, the thirdco-culturing step is repeated (e.g., once, twice or three times).

In some embodiments according to any one of the methods described above,the plurality of tumor antigen peptides is a plurality of synthetictumor antigen peptides. In some embodiments, the plurality of tumorantigen peptides is not obtained from a cell sample.

In some embodiments according to any one of the methods described above,the plurality of tumor antigen peptides comprises general tumor antigenpeptide(s), cancer-type specific antigen peptide(s), and/or neoantigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises one or more neoantigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises (e.g., consists of)neoantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises at least about 5 (e.g., at least about 10, 20, 30, 40or more) different tumor antigen peptides.

In some embodiments according to any one of the methods described above,the target tumor antigen peptide is derived from a tumor antigenselected from the group consisting ofhTERT, p53, Survivin, NY-ESO-1,CEA, CCND1, RGS5, MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3,CDCA1, WT1, KRAS, PARP4, MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6,HPV18-E7, HPV58-E6, HPV58-E7, HBcAg, HBV polymerase, GPC3, SSX, and AFP.

In some embodiments according to any one of the methods described above,the method further comprises identifying a target tumor epitope from thetarget tumor antigen peptide, wherein the target tumor epitope elicitsspecific response by the enriched population of activated T cells, andcontacting a population of APCs with the target tumor epitope to obtainthe population of APCs loaded with the target tumor antigen peptide.

In some embodiments according to any one of the methods described above,the next-generation sequencing is single cell sequencing. In someembodiments, the next-generation sequencing is bulk sequencing. In someembodiments, the sequencing step comprises sequencing a bulk sample ofthe tumor antigen-specific T cells, and single-cell sequencing of aplurality of the tumor antigen-specific T cells. In some embodiments,the sequencing step comprises bulk sequencing of a first portion of thetumor antigen-specific T cells to provide a plurality of genes encodingTCRα and TCRβ, and single-cell sequencing of a second portion of thetumor antigen-specific T cells providing cognate pairing information ofthe plurality of genes encoding TCRα and TCRβ, thereby providing aplurality of TCRs based on paired genes encoding TCRα and TCRβ. In someembodiments, the tumor antigen-specific T cells are stimulated with APCsloaded with the target tumor antigen peptide prior to thenext-generation sequencing.

In some embodiments according to any one of the methods described above,the individual has partial response (PR), complete response (CR), orstable disease (SD) after receiving the MASCT. In some embodiments, theMASCT comprises: co-culturing a population of DCs loaded with aplurality of tumor antigen peptides comprising the target tumor antigenpeptide and a population of T cells to obtain a population of activatedT cells. In some embodiments, the MASCT comprises: (i) co-culturing apopulation of DCs loaded with a plurality of tumor antigen peptidescomprising the target tumor antigen peptide and a population of T cellsin an initial co-culture medium comprising one or more (e.g., aplurality of cytokines) and an immune checkpoint inhibitor to provide aco-culture; and (ii) adding an anti-CD3 antibody to the co-culture atabout 3 to 7 days after the co-culturing starts, thereby obtaining thepopulation of activated T cells. In some embodiments, the MASCTcomprises: (i) contacting a population of DCs with a plurality of tumorantigen peptides comprising the target tumor antigen peptide to obtain apopulation of DCs loaded with the plurality of tumor antigen peptides;and (ii) culturing the population of DCs loaded with the plurality oftumor antigen peptides in a DC maturation medium comprising MPLA. Insome embodiments, the DC maturation medium comprises INFy, MPLA andPGE2. In some embodiments, the MASCT comprises administering to theindividual an effective amount of the DCs loaded with the plurality oftumor antigen peptides. In some embodiments, the individual haspreviously received the MASCT for at least three times.

In some embodiments according to any one of the methods described above,TCRs specifically recognizing a plurality of target tumor antigenpeptides are obtained in parallel.

In some embodiments according to any one of the methods described above,the method further comprises expressing each pair of genes encoding TCRαand TCRβ in a host immune cell to provide an engineered immune cellexpressing a TCR, and assessing response of the engineered immune cellto the target tumor antigen peptide. Further provided is a method ofobtaining a TCR specifically recognizing a target tumor antigen peptideusing the method above, wherein the TCR is selected based on theresponse of the engineered immune cell expressing the TCR to the targettumor antigen peptide. In some embodiments, the method further comprisesdetermining HLA restriction of the TCR. In some embodiments, the TCR hasa HLA haplotype restriction that is predominant in Asians. In someembodiments, the method further comprises affinity maturation of theTCR. In some embodiments, the method further comprises enhancing theparing of the TCRα and TCRβ chains in the TCR. In some embodiments, themethod further comprises enhancing the expression of the TCR. In someembodiments, the target tumor antigen peptide is derived from CEA, RSG-5or HPV 18-E7.

Also provided is a tumor-specific TCR obtained using any one of themethods described above.

Another aspect of the present application provides a tumor-specific TCRcomprising: (a) a TCRα chain comprising a complementary determiningregion (CDR) 3 having at least about 90% sequence identity to any one ofthe amino acid sequences of SEQ ID NOs: 4, 10, and 16; and a TCRβ chaincomprising a CDR3 comprising an amino acid sequence having at leastabout 90% sequence identity to any one of the amino acid sequences ofSEQ ID NOs: 7, 13, and 19; (b) a TCRα chain comprising a complementarydetermining region (CDR) 3 having at least about 90% sequence identityto any one of the amino acid sequences of SEQ ID NOs: 22, 28, 34, 40,46, and 52; and a TCRβ chain comprising a CDR3 comprising an amino acidsequence having at least about 90% sequence identity to any one of theamino acid sequences of SEQ ID NOs: 7, 13, 19, 25, 31, 37, 43, 49, and55; or (c) a TCRα chain comprising a complementary determining region(CDR) 3 having at least about 90% sequence identity to any one of theamino acid sequences of SEQ ID NOs: 58, 64, 70, 76, 87 and 93; and aTCRβ chain comprising a CDR3 comprising an amino acid sequence having atleast about 90% sequence identity to any one of the amino acid sequencesof SEQ ID NOs: 61, 67, 73, 79, 90 and 96.

In some embodiments, the tumor-specific TCR comprises: (a) a TCRα chaincomprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 4,and a TCRβ chain comprising a CDR3 comprising the amino acid sequence ofSEQ ID NO: 7; (b) a TCRα chain comprising a CDR3 comprising the aminoacid sequence of SEQ ID NO: 10, and a TCRβ chain comprising a CDR3comprising the amino acid sequence of SEQ ID NO: 13; (c) a TCRα chaincomprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 16,and a TCRβ chain comprising a CDR3 comprising the amino acid sequence ofSEQ ID NO: 19; (d) a TCRα chain comprising a CDR3 comprising the aminoacid sequence of SEQ ID NO: 22, and a TCRβ chain comprising a CDR3comprising the amino acid sequence of SEQ ID NO: 25; (e) a TCRα chaincomprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 28,and a TCRβ chain comprising a CDR3 comprising the amino acid sequence ofSEQ ID NO: 31; (f) a TCRα chain comprising a CDR3 comprising the aminoacid sequence of SEQ ID NO: 34, and a TCRβ chain comprising a CDR3comprising the amino acid sequence of SEQ ID NO: 37; (g) a TCRα chaincomprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 40,and a TCRβ chain comprising a CDR3 comprising the amino acid sequence ofSEQ ID NO: 43; (h) a TCRα chain comprising a CDR3 comprising the aminoacid sequence of SEQ ID NO: 46, and a TCRβ chain comprising a CDR3comprising the amino acid sequence of SEQ ID NO: 49; (i) a TCRα chaincomprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 52,and a TCRβ chain comprising a CDR3 comprising the amino acid sequence ofSEQ ID NO: 55; (j) a TCRα chain comprising a CDR3 comprising the aminoacid sequence of SEQ ID NO: 58, and a TCRβ chain comprising a CDR3comprising the amino acid sequence of SEQ ID NO: 61; (k) a TCRα chaincomprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 64,and a TCRβ chain comprising a CDR3 comprising the amino acid sequence ofSEQ ID NO: 67; (1) a TCRα chain comprising a CDR3 comprising the aminoacid sequence of SEQ ID NO: 70, and a TCRβ chain comprising a CDR3comprising the amino acid sequence of SEQ ID NO: 73; (m) a TCRα chaincomprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 76,and a TCRβ chain comprising a CDR3 comprising the amino acid sequence ofSEQ ID NO: 79; (n) a TCRα chain comprising a CDR3 comprising the aminoacid sequence of SEQ ID NO: 87, and a TCRβ chain comprising a CDR3comprising the amino acid sequence of SEQ ID NO: 90; or (o) a TCRα chaincomprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 93,and a TCRβ chain comprising a CDR3 comprising the amino acid sequence ofSEQ ID NO: 96.

Further provided is a tumor-specific TCR comprising: (a) a TCRα chaincomprising CDRs of any one of the amino acid sequences of SEQ ID NOs: 5,11 and 17, and a TCRβ chain comprising CDRs of any one of the amino acidsequences of SEQ ID NOs: 8, 14 and 20; (b) a TCRα chain comprising CDRsof any one of the amino acid sequences of SEQ ID NOs: 23, 29, 35, 41, 47and 53, and a TCRβ chain comprising CDRs of any one of the amino acidsequences of SEQ ID NOs: 26, 32, 38, 44, 50 and 56; or (c) a TCRα chaincomprising CDRs of any one of the amino acid sequences of SEQ ID NOs:59, 65, 71, 77, 88 and 94, and a TCRβ chain comprising CDRs of any oneof the amino acid sequences of SEQ ID NOs: 62, 68, 74, 80, 91 and 97.

In some embodiments according to any one of the tumor-specific TCRsdescribed above, the tumor-specific TCR is a human TCR. In someembodiments, the tumor-specific TCR is a chimeric TCR, such as amurinized TCR, e.g., a TCR comprising murine constant regions of TCRαand β chains.

In some embodiments according to any one of the tumor-specific TCRsdescribed above, the tumor-specific TCR comprises: (a) a TCRα chaincomprising an amino acid sequence having at least about 80% identity toany one of the amino acid sequences of SEQ ID NOs: 5, 11 and 17, and aTCRβ chain comprising an amino acid sequence having at least about 80%identity to any one of the amino acid sequences of SEQ ID NOs: 8, 14 and20; (b) a TCRα chain comprising an amino acid sequence having at leastabout 80% identity to any one of the amino acid sequences of SEQ ID NOs:23, 29, 35, 41, 47 and 53, and a TCRβ chain comprising an amino acidsequence having at least about 80% identity to any one of the amino acidsequences of SEQ ID NOs: 26, 32, 38, 44, 50 and 56; or (c) a TCRα chaincomprising an amino acid sequence having at least about 80% identity toany one of the amino acid sequences of SEQ ID NOs: 59, 65, 71, 77, 88and 94, and a TCRβ chain comprising an amino acid sequence having atleast about 80% identity to any one of the amino acid sequences of SEQID NOs: 62, 68, 74, 80, 91 and 97.

Also provided is an isolated nucleic acid encoding the TCRα chain and/orthe TCRβ chain of the tumor-specific TCR according to any one of thetumor-specific TCRs described above, a vector comprising the isolatednucleic acid(s).

One aspect of the present application provides an engineered immune cellcomprising the tumor-specific TCR according to any one of thetumor-specific TCRs, the isolated nucleic acids, or the vectorsdescribed above.

In some embodiments, the immune cell is a T cell. In some embodiments,there is provided a pharmaceutical composition comprising the engineeredimmune cell according to any one of the engineered immune cellsdescribed above, and a pharmaceutically acceptable carrier.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising administering to the individual an effectiveamount of the pharmaceutical composition according to any one of thepharmaceutical compositions described above.

In some embodiments, there is provided a library of tumor-specific TCRsobtained using the method according to any one of the methods describedabove.

Further provided are kits, medicines, and articles of manufacturecomprising any one of the compositions (such as isolated nucleic acids,vectors and engineered immune cells) as described above.

These and other aspects and advantages of the present invention willbecome apparent from the subsequent detailed description and theappended claims. It is to be understood that one, some, or all of theproperties of the various embodiments described herein may be combinedto form other embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overview of an exemplary method for cloning a pluralityof TCRs specifically recognizing a target tumor antigen peptide.

FIG. 2 shows clinical data of a patient with metastatic cervical cancertreated with MASCT. The bottom panel shows ECT results of the patenttaken in December 2013 (prior to any MASCT treatments), in November 2016(after achieving Stable Disease status on MASCT), and in November 2017.The arrows and circles point to the metastasis site on the rightsacroiliac joint bone, showing reduction of the metastatic tumor and noadditional metastasis in response to MASCT treatments.

FIGS. 3A-3B show specific immune response by the patient’s PBMCs againstthe cervical carcinoma antigen peptide pool (pep pool), and each tumorantigen peptide in the pool after customized MASCT treatments asdetermined by ELISPOT. W/O = response without stimulation with anyantigen peptide. ENV refers to experiment with an irrelevant peptide.The dotted line indicates a threshold of no elevated immune response asmeasured by spots per 200,000 cells which reflect IFNy secretion levels.FIG. 3B shows consistently strong immune response by the patient’s PBMCsagainst the HPV18-E7, RGS-5 and CEA peptides.

FIG. 4 shows an exemplary method (“Method 2”) for preparing tumorantigen-specific T cells as described in Example 2. FIG. 5 shows cellproliferation at various time points in the preparation of tumorantigen-specific T cells. FIGS. 6A-6B shows the percentages of IFNy⁺CD3⁺tumor antigen-specific T cells in various co-culture samples.

FIG. 7 shows optimization of the exemplary method of FIG. 4 (“Method2m”) for preparing tumor antigen-specific T cells as described inExample 2. FIG. 8 shows cell proliferation at various time points in thepreparation of tumor antigen-specific T cells. FIGS. 9A-9B show thepercentages of IFNy⁺CD3⁺ tumor antigen-specific T cells in variousco-culture samples. FIG. 9C shows the percentages of IFNγ⁺TNFα⁺ tumorantigen-specific T cells in various co-culture samples.

FIG. 10 shows the percentages of IFNγ⁺CD4⁺ and IFNγ⁺CD8⁺ cells in tumorantigen-specific cells stimulated by each of the tumor antigen peptides.The tumor antigen-specific cells were prepared using PBMCs from apatient who has clinically benefitted from MASCT and a pool of tumorantigen peptides derived from CEA, RGS5 and HPV18-E7. FIG. 11 shows thefrequencies of unique TCR clonotypes in various T cell samplesdetermined by next-generation sequencing.

FIG. 12 summarizes the numbers of unique clonotypes of TCRα and TCRβsequences that may be specific for CEA, RGS5 and HPV18-E7 peptidesrespectively from tumor-antigen specific T cell preparations usingdifferent samples of PBMCs from a patient who has clinically benefittedfrom MASCT. The clonotypes were selected based on the expressionfrequency patterns in the various samples as shown in FIG. 11 . Theresults were obtained by next-generation sequencing of bulk T cellsamples.

FIG. 13 shows sub-pools of fragments of the HPV18-E7, RGS-5 and CEApeptides, which were screened for specific immune response by tumorantigen-specific T cells. The RGS5-OLP5 peptide was shown to elicit thestrongest specific response by the tumor antigen-specific T cellsprepared using Method 2 and Method 2m. FIG. 14A shows percentages ofIFNy⁺CD3⁺ tumor antigen-specific T cells after the tumorantigen-specific T cells prepared using Method 2m were stimulated byeach of the sub-pool of antigen peptide fragments. FIG. 14B showspercentages of IFNγ⁺TNFα⁺ tumor antigen-specific T cells after the tumorantigen-specific T cells prepared using Method 2 were stimulated by eachof the sub-pool of antigen peptide fragments.

FIGS. 15A-15B show exemplary methods for preparing tumorantigen-specific T cells from a frozen stock of tumor antigen-specific Tcells prepared by Method 2 or Method 2m as described in Example 3. FIG.16 shows cell proliferation at various time points in the preparation oftumor antigen-specific T cells. FIG. 17A shows the percentages ofIFNγ⁺CD3⁺ tumor antigen-specific T cells in various co-culture samples.FIG. 17B shows the percentages of IFNγ⁺TNFα⁺ tumor antigen-specific Tcells in various co-culture samples.

FIGS. 18A-18B show the freqzziruencies of unique clonotypes of TCRα andTCRβ in various T cell samples determined by next-generation sequencing.

FIG. 19 shows results of TCRα and TCRβ pairing results using iPairAnalyzer determined by single-cell sequencing. Wells in a 96-well plategiving rise to successfully paired TCRα and TCRβ sequences are shown ingray and marked with the well position number. Wells giving rise to onlyTCRβ sequences are shown in light gray and wells giving rise to onlyTCRα sequences are shown in dark gray.

FIG. 20 shows exemplary methods for preparing tumor antigen-specific Tcells as described in Example 2.

FIG. 21A shows cell proliferation at various time points in thepreparation of tumor antigen-specific T cells. FIG. 21B shows thepercentages of IFNγ+CD3+ tumor antigen-specific T cells before and afterthe enrichment step.

FIGS. 22A-22B show cell proliferation and percentages of tumorantigen-specific T cell populations in various co-culture samples.

FIG. 23 shows exemplary validation steps for tumor-specific TCRs.

FIG. 24 shows exemplary TCR constructs.

FIGS. 25A-25B show compositions and sequences of exemplary TCRconstructs.

FIGS. 26A-26D show validation results of 09B03 and related TCRconstructs. The sequences in FIG. 26B are, from top to bottom: SEQ IDNOs 83, 250, 251, 252, 253, 82 and 82.

FIGS. 27A-27D show validation results of P09E06 and related TCRconstructs. The sequences in FIG. 27B are, from top to bottom: SEQ IDNOs 83, 250, 251, 252, 253, 82 and 82.

FIGS. 28A-28C show validation results of 09B12 and related TCRconstructs. The sequences in FIG. 28B are, from top to bottom: SEQ ID NO86, and aal-15, aa5-19, aa9-23, aa13-27, aa16-30 of SEQ ID NO 86, andSEQ ID NO 85.

FIGS. 29A-29C show validation results of 09E01 and related TCRconstructs.

FIGS. 30A-30D show validation results of 10F04 and related TCRconstructs. The sequences in FIG. 30B are, from top to bottom: SEQ ID NO86, and aal-15, aa5-19, aa9-23, aa13-27, aa16-30 of SEQ ID NO 86, andSEQ ID NO 85.

FIGS. 31A-31C show validation results of 33D05 and related TCRconstructs.

FIGS. 32A-32D show validation results of P09B08 and related TCRconstructs. The sequences in FIG. 32B are, from top to bottom: SEQ ID NO86, and aal-15, aa5-19, aa9-23, aa13-27, aa16-30 of SEQ ID NO 86, andSEQ ID NO 85.

FIGS. 33A-33G show validation results of 09H05 and related TCRconstructs. The sequences in FIGS. 33C and 33D are, from top to bottom:SEQ ID NOs 83, 250, 251, 252, 253, 82 and 82.

FIGS. 34A-34G show validation results of 09D01 and related TCRconstructs. The sequences in FIGS. 34C and 34D are, from top to bottom:SEQ ID NOs 83, 250, 251, 252, 253, 82 and 82.

FIGS. 35A-35G show validation results of 33A02 and related TCRconstructs. The sequences in FIGS. 35C and 35D are, from top to bottom:SEQ ID NO 86, and aal-15, aa5-19, aa9-23, aa13-27, aa16-30, and aa5-19of SEQ ID NO 86.

FIG. 36 shows specific immune response by the patient’s PBMCs againstthe tumor antigen peptide pools, and each tumor antigen peptide in thepool after MASCT treatments as determined by ELISPOT assays. Percentagesindicate reduced peptide concentration. For example, 1/20 base-pepindicates a pool of general tumor antigen peptides at 20 times dilution.

FIG. 37 shows Round 1 protocol of an exemplary two-round method forpreparing tumor antigen-specific T cells using PBMCs from Patient SMZ.

FIG. 38A shows cell proliferation at various time points in Round 1.FIG. 38B shows percentages of IFNy⁺CD3⁺ tumor antigen-specific T cellpopulations after the enrichment step.

FIGS. 39A-39E shows percentages of tumor antigen-specific T cellpopulations in various co-culture samples of Round 1. FIG. 39F showseffector T cell populations in IFNγ⁺CD4⁺ tumor antigen-specific T cellsobtained at the end of Round 1.

FIG. 40 shows Round 2 protocol of an exemplary two-round method forpreparing tumor antigen-specific T cells using PBMCs from Patient SMZ.

FIG. 41A shows number of T cells and tumor-specific T cells at varioustime points in Round 2. FIGS. 41B-41C show percentages of tumorantigen-specific T cell populations in various co-culture samples ofRound 2.

FIG. 42 show number of T cells and tumor antigen-specific T cells atvarious time points of Round 1 and Round 2.

DETAILED DESCRIPTION OF THE INVENTION

The present application provides a platform for cloning a plurality of Tcell receptors (TCRs) specifically recognizing one or more tumor antigenpeptides using PBMCs or T cells from an individual who has clinicallybenefitted from an immunotherapy, such as Multiple Antigen Specific CellTherapy (“MASCT”). The methods described herein comprise enrichment ofactivated T cells from a co-culture of T cells with antigen-loadeddendritic cells (“DCs”), followed by co-culturing of the enrichedactivated T cells with antigen-loaded DCs to provide tumorantigen-specific T cells for bulk and/or single-cell sequencing toobtain a plurality of paired TCRα and TCRβ genes. The source of T cells,as well as the enrichment and co-culturing steps in the methodsdescribed herein contribute to the high percentage (e.g., at least about1% or higher for bulk sequencing of T cell samples, or at least 10% orhigher for single-T cell sequencing) of tumor antigen-specific T cellsthat respond specifically to the target tumor antigen peptide, which isessential for obtaining cognate pairing information for the TCRα andTCRβ genes of predominant TCR clonotypes by next-generation sequencing.Tumor specific TCRs, engineered immune cells expressing the TCRs andmethods of treating cancer using the engineered immune cells are alsoprovided.

TCRs currently under development and in clinical trials in the field aretypically cloned from PBMCs of healthy human donors, which arestimulated with pre-determined tumor antigen epitope peptides. As aresult, clinical response in patients treated with T cells expressingsuch TCRs is unpredictable. In contrast, the TCRs of the presentapplication are obtained from individuals who have clinically benefittedfrom MASCT treatment, indicating anti-tumor potential of TCRs targetingthe tumor antigen epitope(s) contained in the target tumor antigenpeptides.

Additionally, currently known TCRs cloned from PBMCs of healthy humandonors have low affinity to their target tumor antigen epitope-HLAcomplexes. Optimization of the amino acid sequences of the TCRα and TCRβchains are needed to improve the affinity of the TCRs, which increasesthe risk of cross-reaction of the TCRs, off-target side effects andsevere toxicity. In contrast, because the TCRs of the presentapplication are cloned with cancer patients who have demonstratedclinical response, the TCRs described herein may not require affinityoptimization, and thus promises improved safety profile in the clinics.

In some embodiments, TCRs are cloned from individuals of a racial groupin order to provide TCRs having HLA restrictions that reflect thepredominant HLA haplotypes of the racial group. Most of the TCRs inclinical trials today are HLA-restrictive for haplotypes, such asHLA-DPB1*0401 and HLA-A*0201, that are predominant in Caucasianpopulations. The methods described herein may be used to obtain TCRsthat are HLA-restrictive for any a racial group of interest, including,for example, HLA-A*1101 or HLA-A*2402-restrictive TCRs that may be moreefficacious for treatment of Asian patients.

Accordingly, one aspect of the present application provides a method ofobtaining a plurality of T cell receptors (TCRs) specificallyrecognizing a target tumor antigen peptide, comprising: a) a firstco-culturing step comprising co-culturing a first population ofdendritic cells (DCs) loaded with the target tumor antigen peptide witha population of T cells from an individual to obtain a first co-culture;b) an enrichment step comprising subjecting the first co-culture to anenrichment process to obtain enriched activated T cells; c) a secondco-culturing step comprising co-culturing the enriched activated T cellswith a second population of DCs loaded with the target tumor antigenpeptide to obtain a population of tumor antigen-specific T cells,wherein at least about 10% (e.g., at least about 20%, or at least about50%) of the tumor antigen-specific T cells specifically responds to thetarget tumor antigen peptide; and d) a sequencing step, comprisingsubjecting the tumor antigen-specific T cells to next-generationsequencing (e.g., single-cell sequencing) to identify a plurality ofpairs of genes encoding TCRα and TCRβ, thereby providing the pluralityof T cell receptors based on paired genes encoding TCRα and TCRβ;wherein the individual has clinically benefitted from a Multiple AntigenSpecific Cell Therapy (“MASCT”) comprising administering to theindividual an effective amount of activated T cells prepared byco-culturing a population of T cells with a population of dendriticcells loaded with a plurality of tumor antigen peptides comprising thetarget tumor antigen peptide. In some embodiments, TCRs specificallyrecognizing a plurality of target tumor antigens are obtained inparallel in the method.

I. Definitions

Terms are used herein as generally used in the art, unless otherwisedefined as follows.

As used herein, “a plurality of tumor antigen peptides,” “multiple tumorantigen peptides,” “a pool of tumor antigen peptides” and “a tumorantigen peptides pool” are used interchangeably to refer to acombination of two or more tumor antigen peptides.

As used herein, “antigen presenting cells loaded with a plurality oftumor antigen peptides” and “antigen presenting cells loaded with one ormore tumor antigen peptides” are also referred to as “antigen-loadedantigen presenting cells.” Antigen presenting cells (“APCs”) loaded witha plurality of tumor antigen peptides are APCs that have enhancedpresentation of one or more tumor antigen peptides or fragments thereofamong the plurality of tumor antigen peptides. In some embodiments, theantigen-loaded APCs are antigen-loaded DCs. In some embodiments, theantigen-loaded APCs are antigen-loaded PBMCs.

As used herein, “activated T cells” refer to a population of monoclonal(e.g. encoding the same TCR) or polyclonal (e.g. with clones encodingdifferent TCRs) T cells that have T cell receptors that recognize atleast one tumor antigen peptide. Activated T cells may contain one ormore subtypes of T cells, including, but not limited to, cytotoxic Tcells, helper T cells, natural killer T cells, γδ T cells, regulatory Tcells, and memory T cells.

“Tumor antigen-specific T cells” and “tumor specific T cells” are usedherein interchangeably.

As used herein, “T cell receptor” or “TCR” refers to an endogenous orengineered T cell receptor comprising an extracellular antigen bindingdomain that binds to a specific antigen epitope bound in an MHCmolecule. A TCR may comprise a TCRα polypeptide chain and a TCRβpolypeptide chain. “Tumor-specific TCR” refers to a TCR thatspecifically recognizes a tumor antigen expressed by a tumor cell.“TCR-T” refers to a T cell that expresses a recombinant TCR.

As used herein, “immune checkpoint inhibitor” refers to an agent(including an antibody) that inhibits or blocks an inhibitory immunecheckpoint molecule on an immune cell (such as T cell) or a tumor cell.“Immune checkpoint molecules” include molecules that turn up an immunesignal (i.e., “co-stimulatory molecules”), or molecules that turn downan immune signal (i.e., “inhibitory immune checkpoint molecules”)against a tumor cell.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: decreasing one moresymptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the spread (e.g.,metastasis) of the disease, preventing or delaying the occurrence orrecurrence of the disease, delay or slowing the progression of thedisease, ameliorating the disease state, providing a remission (whetherpartial or total) of the disease, decreasing the dose of one or moreother medications required to treat the disease, delaying theprogression of the disease, increasing the quality of life, and/orprolonging survival. Also encompassed by “treatment” is a reduction ofpathological consequence of cancer. The methods of the inventioncontemplate any one or more of these aspects of treatment.

As used herein, “delaying” the development of cancer means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individual being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe disease. A method that “delays” development of cancer is a methodthat reduces probability of disease development in a given time frameand/or reduces the extent of the disease in a given time frame, whencompared to not using the method. Such comparisons are typically basedon clinical studies, using a statistically significant number ofindividuals. Cancer development can be detectable using standardmethods, including, but not limited to, computerized axial tomography(CAT Scan), Magnetic Resonance Imaging (MRI), abdominal ultrasound,clotting tests, arteriography, or biopsy. Development may also refer tocancer progression that may be initially undetectable and includesoccurrence, recurrence, and onset.

The terms “individual,” “subject” and “patient” are used interchangeablyherein to describe a mammal, including humans. An individual includes,but is not limited to, human, bovine, horse, feline, canine, rodent, orprimate. In some embodiments, the individual is human. In someembodiments, an individual suffers from a disease, such as cancer. Insome embodiments, the individual is in need of treatment.

As is understood in the art, an “effective amount” refers to an amountof a composition (e.g. antigen-loaded DCs, activated T cells orengineered immune cells expressing TCRs) sufficient to produce a desiredtherapeutic outcome (e.g., reducing the severity or duration of,stabilizing the severity of, or eliminating one or more symptoms ofcancer). For therapeutic use, beneficial or desired results include,e.g., decreasing one or more symptoms resulting from the disease(biochemical, histologic and/or behavioral), including its complicationsand intermediate pathological phenotypes presented during development ofthe disease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, enhancing effect of another medication, delaying theprogression of the disease, and/or prolonging survival of patients.

“Adjuvant setting” refers to a clinical setting in which an individualhas had a history of cancer, and generally (but not necessarily) beenresponsive to therapy, which includes, but is not limited to, surgery(e.g., surgery resection), radiotherapy, and chemotherapy. However,because of their history of cancer, these individuals are considered atrisk of development of the disease. Treatment or administration in the“adjuvant setting” refers to a subsequent mode of treatment. The degreeof risk (e.g., when an individual in the adjuvant setting is consideredas “high risk” or “low risk”) depends upon several factors, most usuallythe extent of disease when first treated.

“Neoadjuvant setting” refers to a clinical setting in which the methodis carried out before the primary/definitive therapy.

As used herein, “combination therapy” means that a first agent isadministered in conjunction with another agent. “In conjunction with”refers to administration of one treatment modality in addition toanother treatment modality, such as administration of a compositiondescribed herein (e.g. antigen-loaded DCs, activated T cells orengineered immune cells expressing TCRs) in addition to administrationof another agent (such as an immune checkpoint inhibitor) to the sameindividual. As such, “in conjunction with” refers to administration ofone treatment modality before, during, or after delivery of the othertreatment modality to the individual. Such combinations are consideredto be part of a single treatment regimen or regime.

The term “simultaneous administration,” as used herein, means that afirst therapy and second therapy in a combination therapy areadministered with a time separation of no more than about 15 minutes,such as no more than about any of 10, 5, or 1 minutes. When the firstand second therapies are administered simultaneously, the first andsecond therapies may be contained in the same composition (e.g., acomposition comprising both a first and second therapy) or in separatecompositions (e.g., a first therapy in one composition and a secondtherapy is contained in another composition).

As used herein, the term “sequential administration” means that thefirst therapy and second therapy in a combination therapy areadministered with a time separation of more than about 15 minutes, suchas more than about any of 20, 30, 40, 50, 60, or more minutes. Eitherthe first therapy or the second therapy may be administered first. Thefirst and second therapies are contained in separate compositions, whichmay be contained in the same or different packages or kits.

As used herein, the term “concurrent administration” means that theadministration of the first therapy and that of a second therapy in acombination therapy overlap with each other.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to an individual without causingany significant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

The following definitions may be used to evaluate response based ontarget lesions: “complete response” or “CR” refers to disappearance ofall target lesions; “partial response” or “PR” refers to at least a 30%decrease in the sum of the longest diameters (SLD) of target lesions,taking as reference the baseline SLD; “stable disease” or “SD” refers toneither sufficient shrinkage of target lesions to qualify for PR, norsufficient increase to qualify for PD, taking as reference the nadir SLDsince the treatment started; and “progressive disease” or “PD” refers toat least a 20% increase in the SLD of target lesions, taking asreference the nadir SLD recorded since the treatment started, or, thepresence of one or more new lesions.

The following definitions of response assessments may be used toevaluate a non-target lesion: “complete response” or “CR” refers todisappearance of all non-target lesions; “stable disease” or “SD” refersto the persistence of one or more non-target lesions not qualifying forCR or PD; and “progressive disease” or “PD” refers to the “unequivocalprogression” of existing non-target lesion(s) or appearance of one ormore new lesion(s) is considered progressive disease (if PD for theindividual is to be assessed for a time point based solely on theprogression of non-target lesion(s), then additional criteria arerequired to be fulfilled.

As used herein, the terms “cell”, “cell line”, and “cell culture” areused interchangeably and all such designations include progeny. It isunderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological activity as the original cellsare included.

The term “peptide” refers to a polymer of amino acids no more than about100 amino acids (including fragments of a protein), which may be linearor branched, comprise modified amino acids, and/or be interrupted bynon-amino acids. The term also encompasses an amino acid polymer thathas been modified naturally or by intervention, including, for example,disulfide bond formation, glycosylation, lipidation, acetylation,phosphorylation, or any other manipulation or modification. Alsoincluded within this term are, for example, peptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), as well as other modifications known in the art. Thepeptides described herein may be naturally-occurring, i.e., obtained orderived from a natural source (e.g., blood) or synthesized (e.g.,chemically synthesized or by synthesized by recombinant DNA techniques).

The term “antibody” used herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), multispecific antibodies (e.g., bispecificantibodies), and antibody fragments so long as they exhibit the desiredbiological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

As use herein, the term “specifically binds to,” “recognizes,”“specifically recognizes,” “targets,” or is “specific for” refers tomeasurable and reproducible interactions such as binding between atarget and an antibody, or a receptor and a ligand, or a receptor and anepitope/MHC complex, which is determinative of the presence of thetarget in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, a TCR that binds to orspecifically binds to a target epitope is a TCR that binds the targetepitope/MHC complex with greater affinity, avidity, more readily, and/orwith greater duration than it binds to other epitope/MHC complexes. Inone embodiment, the extent of binding of a TCR to an unrelatedepitope/MHC complex is less than about 10% of the binding of the TCR tothe target epitope/MHC complex as measured, e.g., by a radioimmunoassay(RIA). In certain embodiments, a TCR that specifically binds to a targetepitope (i.e., target epitope/MHC complex) has a dissociation constant(Kd) of ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. In certainembodiments, a TCR specifically binds to an epitope on a protein that isconserved among the protein from different species. In anotherembodiment, specific binding can include, but does not require exclusivebinding.

The term “isolated nucleic acid” as used herein is intended to mean anucleic acid of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated nucleic acid” (1)is not associated with all or a portion of a polynucleotide in which the“isolated nucleic acid” is found in nature, (2) is operably linked to apolynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

It is understood that aspect and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

The term “about X-Y” used herein has the same meaning as “about X toabout Y.”

As used herein, reference to “not” a value or parameter generally meansand describes “other than” a value or parameter. For example, the methodis not used to treat cancer of type X means the method is used to treatcancer of types other than X.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise.

II. Methods of Obtaining Tumor-Specific TCRs

The present application provides methods of obtaining a plurality of Tcell receptors (TCRs) specifically recognizing one or more target tumorantigen peptides from PBMCs or T cells of an individual that hasclinically benefitted from an immunotherapy. In some embodiments, theimmunotherapy is adoptive T cell therapy comprising administering to theindividual an effective amount of activated T cells that specificallyrecognizes the target tumor antigen peptides or fragments thereof. Insome embodiments, the immunotherapy is Multiple Antigen Specific CellTherapy (“MASCT”) comprising administering to the individual aneffective amount of activated T cells prepared by co-culturing apopulation of T cells with a population of dendritic cells loaded with aplurality of tumor antigen peptides comprising one or more target tumorantigen peptides. In some embodiments, TCRs specifically recognizing asingle target tumor antigen peptide or a fragment thereof (e.g., atarget tumor epitope) are obtained. In some embodiments, TCRsspecifically recognizing a plurality of target tumor antigen peptidesare obtained simultaneously using the method.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: subjecting a population of tumor antigen-specific T cells tonext-generation sequencing (e.g., single-cell sequencing) to identify aplurality of pairs of genes encoding TCRα and TCRβ, thereby providingthe plurality of T cell receptors based on paired genes encoding TCRαand TCRβ; wherein at least about 10% (e.g., at least about 20%, or atleast about 50%) of the tumor antigen-specific T cells specificallyresponds to the target tumor antigen peptide; wherein the population oftumor antigen-specific T cells is prepared by: i) a first co-culturingstep comprising co-culturing a first population of DCs loaded with thetarget tumor antigen peptide with a population of T cells from anindividual to obtain a first co-culture; ii) an enrichment stepcomprising subjecting the first co-culture to an enrichment process toobtain enriched activated T cells; and iii) a second co-culturing stepcomprising co-culturing the enriched activated T cells with a secondpopulation of DCs loaded with the target tumor antigen peptide to obtaina population of tumor antigen-specific T cells; and wherein theindividual has clinically benefitted from a Multiple Antigen SpecificCell Therapy (“MASCT”) comprising administering to the individual aneffective amount of activated T cells prepared by co-culturing apopulation of T cells with a population of DCs loaded with a pluralityof tumor antigen peptides comprising the target tumor antigen peptide.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) co-culturing an enriched population of activated T cellswith a second population of DCs loaded with the target tumor antigenpeptide to obtain a population of tumor antigen-specific T cells,wherein at least about 10% (e.g., at least about 20%, or at least about50%) of the tumor antigen-specific T cells specifically responds to thetarget tumor antigen peptide; and b) subjecting the tumorantigen-specific T cells to next-generation sequencing (e.g.,single-cell sequencing) to identify a plurality of pairs of genesencoding TCRα and TCRβ, thereby providing the plurality of T cellreceptors based on paired genes encoding TCRα and TCRβ; wherein theenriched population of activated T cells is prepared by: i) a firstco-culturing step comprising co-culturing a first population of DCsloaded with the target tumor antigen peptide with a population of Tcells from an individual to obtain a first co-culture; and ii) anenrichment step comprising subjecting the first co-culture to anenrichment process to obtain enriched activated T cells; and wherein theindividual has clinically benefitted from a Multiple Antigen SpecificCell Therapy (“MASCT”) comprising administering to the individual aneffective amount of activated T cells prepared by co-culturing apopulation of T cells with a population of DCs loaded with a pluralityof tumor antigen peptides comprising the target tumor antigen peptide.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) subjecting the a first co-culture comprising a firstpopulation of DCs loaded with the target tumor antigen peptide and apopulation of T cells from an individual to an enrichment process toobtain enriched activated T cells; b) co-culturing the enrichedactivated T cells with a second population of DCs loaded with the targettumor antigen peptide to obtain a population of tumor antigen-specific Tcells, wherein at least about 10% (e.g., at least about 20%, or at leastabout 50%) of the tumor antigen-specific T cells specifically respondsto the target tumor antigen peptide; and c) subjecting the tumorantigen-specific T cells to next-generation sequencing (e.g.,single-cell sequencing) to identify a plurality of pairs of genesencoding TCRα and TCRβ, thereby providing the plurality of T cellreceptors based on paired genes encoding TCRα and TCRβ; and wherein theindividual has clinically benefitted from a Multiple Antigen SpecificCell Therapy (“MASCT”) comprising administering to the individual aneffective amount of activated T cells prepared by co-culturing apopulation of T cells with a population of DCs loaded with a pluralityof tumor antigen peptides comprising the target tumor antigen peptide.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) a first co-culturing step comprising co-culturing a firstpopulation of DCs loaded with the target tumor antigen peptide with apopulation of T cells from an individual to obtain a first co-culture;b) an enrichment step comprising subjecting the first co-culture to anenrichment process to obtain enriched activated T cells; c) a secondco-culturing step comprising co-culturing the enriched activated T cellswith a second population of DCs loaded with the target tumor antigenpeptide to obtain a population of tumor antigen-specific T cells,wherein at least about 10% (e.g., at least about 20%, or at least about50%) of the tumor antigen-specific T cells specifically responds to thetarget tumor antigen peptide; and d) a sequencing step, comprisingsubjecting the tumor antigen-specific T cells to next-generationsequencing (e.g., single-cell sequencing) to identify a plurality ofpairs of genes encoding TCRα and TCRβ, thereby providing the pluralityof T cell receptors based on paired genes encoding TCRα and TCRβ;wherein the individual has clinically benefitted from a Multiple AntigenSpecific Cell Therapy (“MASCT”) comprising administering to theindividual an effective amount of activated T cells prepared byco-culturing a population of T cells with a population of DCs loadedwith a plurality of tumor antigen peptides comprising the target tumorantigen peptide. In some embodiments, the first co-culturing step iscarried out for about 1 to about 3 days prior to the enrichment step. Insome embodiments, the ratio between the population of T cells to thefirst population of antigen-loaded DCs is no more than about 30:1 (e.g.,about 20:1, 15:1 or 10:1). In some embodiments, the population of Tcells in the first co-culturing step is present in PBMCs. In someembodiments, the first population of DCs loaded with the target tumorantigen peptide and the population of T cells are co-cultured in a firstco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines, e.g., IL-2, IL-7, IL-15 and IL-21) and an immunecheckpoint inhibitor (e.g., anti-PD-1 antibody, such as SHR-1210). Insome embodiments, the first co-culture medium comprises IL-2 and ananti-PD-1 antibody. In some embodiments, the ratio between the enrichedpopulation of activated T cells and the second population of DCs loadedwith the target tumor antigen peptide is about 1:1 to about 20:1 (e.g.,about 1:1, 2:1 or 4:1). In some embodiments, the enriched population ofactivated T cells and the second population of DCs loaded with thetarget tumor antigen peptide are co-cultured for about 12 to 25 days. Insome embodiments, the individual has response (PR), complete response(CR), or stable disease (SD) for at least about 6 months (e.g., at leastabout 1 year, 2 years, or more) after receiving the MASCT.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) a first co-culturing step comprising co-culturing a firstpopulation of DCs loaded with the target tumor antigen peptide with apopulation of T cells from an individual to obtain a first co-culture;b) an enrichment step comprising contacting the first co-culture withAPCs (e.g., DCs or PBMCs) loaded with the target tumor antigen peptideto obtain a stimulated co-culture, and isolating from the stimulatedco-culture an enriched population of activated T cells using a ligandthat specifically recognizes a cytokine or cell surface molecule; c) asecond co-culturing step comprising co-culturing the enriched activatedT cells with a second population of DCs loaded with the target tumorantigen peptide to obtain a population of tumor antigen-specific Tcells, wherein at least about 10% (e.g., at least about 20%, or at leastabout 50%) of the tumor antigen-specific T cells specifically respondsto the target tumor antigen peptide; and d) a sequencing step,comprising subjecting the tumor antigen-specific T cells tonext-generation sequencing (e.g., single-cell sequencing) to identify aplurality of pairs of genes encoding TCRα and TCRβ, thereby providingthe plurality of T cell receptors based on paired genes encoding TCRαand TCRβ; wherein the individual has clinically benefitted from aMultiple Antigen Specific Cell Therapy (“MASCT”) comprisingadministering to the individual an effective amount of activated T cellsprepared by co-culturing a population of T cells with a population ofDCs loaded with a plurality of tumor antigen peptides comprising thetarget tumor antigen peptide. In some embodiments, the enrichment stepcomprises contacting the first co-culture with APCs (e.g., DCs or PBMCs)loaded with the target tumor antigen peptide to obtain a stimulatedco-culture, and isolating from the stimulated co-culture an enrichedpopulation of activated T cells using a ligand that specificallyrecognizes IFNy. In some embodiments, the first co-culturing step iscarried out for about 1 to about 3 days prior to the enrichment step. Insome embodiments, the ratio between the population of T cells to thefirst population of antigen-loaded DCs is no more than about 30:1 (e.g.,about 20:1, 15:1 or 10:1). In some embodiments, the population of Tcells in the first co-culturing step is present in PBMCs. In someembodiments, the first population of DCs loaded with the target tumorantigen peptide and the population of T cells are co-cultured in a firstco-culture medium comprising a plurality of cytokines (e.g., IL-2, IL-7,IL-15 and IL-21) and an immune checkpoint inhibitor (e.g., anti-PD-1antibody, such as SHR-1210). In some embodiments, the ratio between theenriched population of activated T cells and the second population ofDCs loaded with the target tumor antigen peptide is about 1:1 to about20:1 (e.g., about 1:1, 2:1 or 4:1). In some embodiments, the enrichedpopulation of activated T cells and the second population of DCs loadedwith the target tumor antigen peptide are co-cultured for about 12 to 25days. In some embodiments, the individual has response (PR), completeresponse (CR), or stable disease (SD) after receiving the MASCT.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) a first co-culturing step comprising co-culturing a firstpopulation of DCs loaded with the target tumor antigen peptide with apopulation of T cells from an individual to obtain a first co-culture;b) an enrichment step comprising subjecting the first co-culture to anenrichment process to obtain enriched activated T cells; c) a secondco-culturing step co-culturing a second population of DCs loaded withthe target tumor antigen peptide with the enriched population ofactivated T cells in an initial second co-culture medium comprising animmune checkpoint inhibitor and optionally one or more cytokines (e.g.,a plurality of cytokines) to provide a second co-culture; and adding ananti-CD3 (e.g., OKT-3) antibody and optionally one or more cytokines tothe second co-culture to obtain a population of tumor antigen-specific Tcells, wherein at least about 10% (e.g., at least about 20%, or at leastabout 50%) of the tumor antigen-specific T cells specifically respondsto the target tumor antigen peptide; and d) a sequencing step,comprising subjecting the tumor antigen-specific T cells tonext-generation sequencing (e.g., single-cell sequencing) to identify aplurality of pairs of genes encoding TCRα and TCRβ, thereby providingthe plurality of T cell receptors based on paired genes encoding TCRαand TCRβ; wherein the individual has clinically benefitted from aMultiple Antigen Specific Cell Therapy (“MASCT”) comprisingadministering to the individual an effective amount of activated T cellsprepared by co-culturing a population of T cells with a population ofDCs loaded with a plurality of tumor antigen peptides comprising thetarget tumor antigen peptide. In some embodiments, the firstco-culturing step is carried out for about 1 to about 3 days prior tothe enrichment step. In some embodiments, the ratio between thepopulation of T cells to the first population of antigen-loaded DCs isno more than about 30:1 (e.g., about 20:1, 15:1 or 10:1). In someembodiments, the population of T cells in the first co-culturing step ispresent in PBMCs. In some embodiments, the first population of DCsloaded with the target tumor antigen peptide and the population of Tcells are co-cultured in a first co-culture medium comprising one ormore cytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2,IL-7, IL-15 and IL-21) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody, such as SHR-1210). In some embodiments, theenrichment step comprises contacting the first co-culture with APCs(e.g., DCs or PBMCs) loaded with the target tumor antigen peptide toobtain a stimulated co-culture, and isolating from the stimulatedco-culture an enriched population of activated T cells using a ligandthat specifically recognizes a cytokine (e.g., IFNy) or a cell surfacemolecule. In some embodiments, the ratio between the enriched populationof activated T cells and the second population of DCs loaded with thetarget tumor antigen peptide is about 1:1 to about 20:1 (e.g., about1:1, 2:1 or 4:1). In some embodiments, the enriched population ofactivated T cells and the second population of DCs loaded with thetarget tumor antigen peptide are co-cultured for about 12 to 25 days. Insome embodiments, the anti-CD3 antibody is added to the secondco-culture no more than about 3 days (e.g., about 2 days) after thesecond co-culturing step starts. In some embodiments, the initial secondco-culture medium comprises IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1antibody. In some embodiments, the individual has response (PR),complete response (CR), or stable disease (SD) after receiving theMASCT.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) a first co-culturing step comprising co-culturing a firstpopulation of DCs loaded with the target tumor antigen peptide with apopulation of T cells from an individual to obtain a first co-culture;b) an enrichment step comprising subjecting the first co-culture to anenrichment process to obtain enriched activated T cells; c) a secondco-culturing step comprising co-culturing the enriched activated T cellswith a second population of DCs loaded with the target tumor antigenpeptide to obtain a population of tumor antigen-specific T cells; d) athird co-culturing step comprising co-culturing a population of thetumor antigen-specific T cells with a population of APCs (e.g., PBMCs,DCs, or cell line APCs) loaded with target tumor antigen peptide toobtain a second population of tumor antigen-specific T cells, wherein atleast about 10% (e.g., at least about 20%, or at least about 50%) of thetumor antigen-specific T cells specifically responds to the target tumorantigen peptide; and e) a sequencing step, comprising subjecting thetumor antigen-specific T cells to next-generation sequencing (e.g.,single-cell sequencing) to identify a plurality of pairs of genesencoding TCRα and TCRβ, thereby providing the plurality of T cellreceptors based on paired genes encoding TCRα and TCRβ; wherein theindividual has clinically benefitted from a Multiple Antigen SpecificCell Therapy (“MASCT”) comprising administering to the individual aneffective amount of activated T cells prepared by co-culturing apopulation of T cells with a population of DCs loaded with a pluralityof tumor antigen peptides comprising the target tumor antigen peptide.In some embodiments, the first co-culturing step is carried out forabout 1 to about 3 days prior to the enrichment step. In someembodiments, the ratio between the population of T cells to the firstpopulation of antigen-loaded DCs is no more than about 30:1 (e.g., about20:1, 15:1 or 10:1). In some embodiments, the population of T cells inthe first co-culturing step is present in PBMCs. In some embodiments,the first population of DCs loaded with the target tumor antigen peptideand the population of T cells are co-cultured in a first co-culturemedium comprising one or more cytokines (such as IL-2 or a plurality ofcytokines, e.g., IL-2, IL-7, IL-15 and IL-21) and an immune checkpointinhibitor (e.g., anti-PD-1 antibody, such as SHR-1210). In someembodiments, the enrichment step comprises contacting the firstco-culture with APCs (e.g., DCs or PBMCs) loaded with the target tumorantigen peptide to obtain a stimulated co-culture, and isolating fromthe stimulated co-culture an enriched population of activated T cellsusing a ligand that specifically recognizes a cytokine (e.g., IFNy) or acell surface molecule. In some embodiments, the ratio between theenriched population of activated T cells and the second population ofDCs loaded with the target tumor antigen peptide is about 1:1 to about20:1 (e.g., about 1:1, 2:1 or 4:1). In some embodiments, the enrichedpopulation of activated T cells and the second population of DCs loadedwith the target tumor antigen peptide are co-cultured for about 12 to 25days. In some embodiments, the second co-culturing step comprisesco-culturing the second population of DCs loaded with the target tumorantigen peptide with the enriched population of activated T cells in aninitial second co-culture medium comprising an immune checkpointinhibitor and optionally one or more cytokines (e.g., a plurality ofcytokines) to provide a second co-culture; and adding an anti-CD3antibody (e.g., OKT-3) and optionally one or more cytokines to thesecond co-culture to obtain a population of tumor antigen-specific Tcells. In some embodiments, the anti-CD3 antibody is added to the secondco-culture no more than about 3 days (e.g., about 2 days) after thesecond co-culturing step starts. In some embodiments, the initial secondco-culture medium comprises IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1antibody. In some embodiments, the ratio between the population of tumorantigen-specific T cells and the population of APCs loaded with thetarget tumor antigen peptide is about 1:1 to about 20:1 (e.g., about4:1). In some embodiments, the population of tumor antigen-specific Tcells and the population of APCs loaded with the target tumor antigenpeptide are co-cultured for about 5 to 9 days (e.g., about 7 days). Insome embodiments, the population of tumor antigen-specific T cells andthe population of APCs loaded with the target tumor antigen peptide areco-cultured in a third co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, thethird co-culturing step is repeated (e.g., once or twice). In someembodiments, the individual has response (PR), complete response (CR),or stable disease (SD) after receiving the MASCT.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) a first co-culturing step comprising co-culturing a firstpopulation of DCs loaded with the target tumor antigen peptide with apopulation of T cells from an individual to obtain a first co-culture;b) an enrichment step comprising contacting the first co-culture withAPCs (e.g., PBMCs or DCs) loaded with the target tumor antigen peptideto obtain a stimulated co-culture, and isolating from the stimulatedco-culture an enriched population of activated T cells using a ligandthat specifically recognizes a cytokine (e.g., IFNy); c) a secondco-culturing step comprising co-culturing a second population of DCsloaded with the target tumor antigen peptide with the enrichedpopulation of activated T cells in an initial second co-culture mediumcomprising an immune checkpoint inhibitor and optionally one or morecytokines (e.g., a plurality of cytokines) to provide a secondco-culture; and adding an anti-CD3 antibody (e.g., OKT-3) and optionallyone or more cytokines to the second co-culture to obtain a population oftumor antigen-specific T cells.; d) a third co-culturing step comprisingco-culturing a population of the tumor antigen-specific T cells with afirst population of APCs (e.g., PBMCs, DCs, or cell line APCs) loadedwith target tumor antigen peptide for about 5 to 9 days (e.g., 7 days),adding to the third co-culture a second population of APCs (e.g., PBMCs,DCs, or cell line APCs) loaded with target tumor antigen peptide andculturing for about 5 to 9 days (e.g., 7 days), and adding to theco-culture a third population of APCs (e.g., PBMCs, DCs, or cell lineAPCs) loaded with target tumor antigen peptide and culturing for about 5to 9 days (e.g., 7 days) to obtain a second population of tumorantigen-specific T cells, wherein at least about 10% (e.g., at leastabout 20%, or at least about 50%) of the tumor antigen-specific T cellsspecifically responds to the target tumor antigen peptide; and e) asequencing step, comprising subjecting the tumor antigen-specific Tcells to next-generation sequencing (e.g., single-cell sequencing) toidentify a plurality of pairs of genes encoding TCRα and TCRβ, therebyproviding the plurality of T cell receptors based on paired genesencoding TCRα and TCRβ; wherein the individual has clinically benefittedfrom a Multiple Antigen Specific Cell Therapy (“MASCT”) comprisingadministering to the individual an effective amount of activated T cellsprepared by co-culturing a population of T cells with a population ofDCs loaded with a plurality of tumor antigen peptides comprising thetarget tumor antigen peptide. In some embodiments, the firstco-culturing step is carried out for about 1 to about 3 days prior tothe enrichment step. In some embodiments, the ratio between thepopulation of T cells to the first population of antigen-loaded DCs isno more than about 30:1 (e.g., about 20:1, 15:1 or 10:1). In someembodiments, the population of T cells in the first co-culturing step ispresent in PBMCs. In some embodiments, the first population of DCsloaded with the target tumor antigen peptide and the population of Tcells are co-cultured in a first co-culture medium comprising one ormore cytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2,IL-7, IL-15 and IL-21) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody, such as SHR-1210). In some embodiments, the ratiobetween the enriched population of activated T cells and the secondpopulation of DCs loaded with the target tumor antigen peptide is about1:1 to about 20:1 (e.g., about 1:1, 2:1 or 4:1). In some embodiments,the enriched population of activated T cells and the second populationof DCs loaded with the target tumor antigen peptide are co-cultured forabout 12 to 25 days. In some embodiments, the anti-CD3 antibody is addedto the second co-culture no more than about 3 days (e.g., about 2 days)after the second co-culturing step starts. In some embodiments, theinitial second co-culture medium comprises IL-2, IL-7, IL-15 and IL-21and an anti-PD-1 antibody. In some embodiments, the ratio between thepopulation of tumor antigen-specific T cells and the first population ofAPCs loaded with the target tumor antigen peptide is about 1:1 to about20:1 (e.g., about 4:1). In some embodiments, the population of tumorantigen-specific T cells and the APCs loaded with the target tumorantigen peptide are co-cultured in a third co-culture medium comprisingone or more cytokines (such as IL-2 or a plurality of cytokines, e.g.,IL-2, IL-7, IL-15) and an anti-CD3 antibody (e.g., OKT3). In someembodiments, the individual has response (PR), complete response (CR),or stable disease (SD) after receiving the MASCT.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) a first co-culturing step comprising co-culturing a firstpopulation of DCs loaded with the target tumor antigen peptide with apopulation of T cells from an individual to obtain a first co-culture;b) an enrichment step comprising subjecting the first co-culture to anenrichment process to obtain enriched activated T cells; c) a secondco-culturing step comprising co-culturing the enriched activated T cellswith a second population of DCs loaded with the target tumor antigenpeptide to obtain a population of tumor antigen-specific T cells; d) ascreening step comprising identifying a target tumor epitope from thetarget tumor antigen peptide, wherein the target tumor epitope elicitsspecific response by the enriched population of activated T cells, andcontacting a population of APCs (e.g., PBMCs, DCs, or cell line APCs)with the target tumor epitope to obtain a population of APCs loaded withthe target tumor antigen peptide; e) a third co-culturing stepcomprising co-culturing a population of the tumor antigen-specific Tcells with the population of APCs loaded with target tumor antigenpeptide to obtain a second population of tumor antigen-specific T cells,wherein at least about 10% (e.g., at least about 20%, or at least about50%) of the tumor antigen-specific T cells specifically responds to thetarget tumor antigen peptide; and f) a sequencing step, comprisingsubjecting the tumor antigen-specific T cells to next-generationsequencing (e.g., single-cell sequencing) to identify a plurality ofpairs of genes encoding TCRα and TCRβ, thereby providing the pluralityof T cell receptors based on paired genes encoding TCRα and TCRβ;wherein the individual has clinically benefitted from a Multiple AntigenSpecific Cell Therapy (“MASCT”) comprising administering to theindividual an effective amount of activated T cells prepared byco-culturing a population of T cells with a population of DCs loadedwith a plurality of tumor antigen peptides comprising the target tumorantigen peptide. In some embodiments, the first co-culturing step iscarried out for about 1 to about 3 days prior to the enrichment step. Insome embodiments, the ratio between the population of T cells to thefirst population of antigen-loaded DCs is no more than about 30:1 (e.g.,about 20:1, 15:1 or 10:1). In some embodiments, the population of Tcells in the first co-culturing step is present in PBMCs. In someembodiments, the first population of DCs loaded with the target tumorantigen peptide and the population of T cells are co-cultured in a firstco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines, e.g., IL-2, IL-7, IL-15 and IL-21) and an immunecheckpoint inhibitor (e.g., anti-PD-1 antibody, such as SHR-1210). Insome embodiments, the enrichment step comprises contacting the firstco-culture with APCs (e.g., DCs or PBMCs) loaded with the target tumorantigen peptide to obtain a stimulated co-culture, and isolating fromthe stimulated co-culture an enriched population of activated T cellsusing a ligand that specifically recognizes a cytokine (e.g., IFNy) or acell surface molecule. In some embodiments, the ratio between theenriched population of activated T cells and the second population ofDCs loaded with the target tumor antigen peptide is about 1:1 to about20:1 (e.g., about 1:1, 2:1 or 4:1). In some embodiments, the enrichedpopulation of activated T cells and the second population of DCs loadedwith the target tumor antigen peptide are co-cultured for about 12 to 25days. In some embodiments, the second co-culturing step comprisesco-culturing the second population of DCs loaded with the target tumorantigen peptide with the enriched population of activated T cells in aninitial second co-culture medium comprising an immune checkpointinhibitor and optionally one or more cytokines (e.g., a plurality ofcytokines) to provide a second co-culture; and adding an anti-CD3antibody (e.g., OKT-3) and optionally one or more cytokines to thesecond co-culture to obtain a population of tumor antigen-specific Tcells. In some embodiments, the anti-CD3 antibody is added to the secondco-culture no more than about 3 days (e.g., about 2 days) after thesecond co-culturing step starts. In some embodiments, the initial secondco-culture medium comprises IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1antibody. In some embodiments, the ratio between the population of tumorantigen-specific T cells and the population of APCs loaded with thetarget tumor antigen peptide is about 1:1 to about 20:1 (e.g., about4:1). In some embodiments, the population of tumor antigen-specific Tcells and the population of APCs loaded with the target tumor antigenpeptide are co-cultured for about 5 to 9 days (e.g., about 7 days). Insome embodiments, the population of tumor antigen-specific T cells andthe population of APCs loaded with the target tumor antigen peptide areco-cultured in a third co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, thethird co-culturing step is repeated (e.g., once or twice). In someembodiments, the individual has response (PR), complete response (CR),or stable disease (SD) after receiving the MASCT.

Any one of the methods described herein may be used to obtain TCRsspecifically recognizing a plurality of target tumor antigen peptides inparallel by using APCs (e.g., PBMCs or DCs) loaded with a plurality oftarget tumor antigen peptides in the first co-culturing step, theenrichment step and the second co-culturing step. In some embodiments,APCs (e.g., PBMCs, DCs or cell line APCs) loaded with individual targettumor antigen peptides are used in the third co-culturing step toprepare individual populations of tumor antigen-specific T cells forsequencing. In some embodiments, APCs (e.g., PBMCs, DCs or cell lineAPCs) loaded with the plurality of target tumor antigen peptides areused in the third co-culturing step to prepare a pooled population oftumor antigen-specific T cells for sequencing. In some embodiments, TCRsspecifically recognizing at least about any one of 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, or more target tumor antigen peptides are obtainedusing the method.

Thus, in some embodiments, there is provided a method of obtaining aplurality of TCRs specifically recognizing a plurality of target tumorantigen peptides, comprising: a) a first co-culturing step comprisingco-culturing a first population of DCs loaded with the plurality oftarget tumor antigen peptides with a population of T cells from anindividual to obtain a first co-culture; b) an enrichment stepcomprising subjecting the co-culture to an enrichment process to obtainenriched activated T cells; c) a second co-culturing step comprisingco-culturing the enriched activated T cells with a second population ofDCs loaded with the plurality of target tumor antigen peptides to obtaina population of tumor antigen-specific T cells, wherein at least about10% (e.g., at least about 20%, or at least about 50%) of the tumorantigen-specific T cells specifically responds to one or more targettumor antigen peptides from the plurality of target tumor antigenpeptides; and d) a sequencing step, comprising subjecting the tumorantigen-specific T cells to next-generation sequencing (e.g.,single-cell sequencing) to identify a plurality of pairs of genesencoding TCRα and TCRβ, thereby providing the plurality of T cellreceptors based on paired genes encoding TCRα and TCRβ; wherein theindividual has clinically benefitted from a Multiple Antigen SpecificCell Therapy (“MASCT”) comprising administering to the individual aneffective amount of activated T cells prepared by co-culturing apopulation of T cells with a population of DCs loaded with a pluralityof tumor antigen peptides comprising the target tumor antigen peptides.In some embodiments, the first co-culturing step is carried out forabout 1 to about 3 days prior to the enrichment step. In someembodiments, the ratio between the population of T cells to the firstpopulation of antigen-loaded DCs is no more than about 30:1 (e.g., about20:1, 15:1 or 10:1). In some embodiments, the population of T cells inthe first co-culturing step is present in PBMCs. In some embodiments,the first population of antigen-loaded DCs and the population of T cellsare co-cultured in a first co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15 and IL-21) and an immune checkpoint inhibitor (e.g., anti-PD-1antibody, such as SHR-1210). In some embodiments, the enrichment stepcomprises contacting the first co-culture with APCs (e.g., DCs or PBMCs)loaded with the plurality of target tumor antigen peptides to obtain astimulated co-culture, and isolating from the stimulated co-culture anenriched population of activated T cells using a ligand thatspecifically recognizes a cytokine (e.g., IFNy) or a cell surfacemolecule. In some embodiments, the ratio between the enriched populationof activated T cells and the second population of antigen-loaded DCs isabout 1:1 to about 20:1 (e.g., about 1:1, 2:1 or 4:1). In someembodiments, the enriched population of activated T cells and the secondpopulation of antigen-loaded DCs are co-cultured for about 12 to 25days. In some embodiments, the second co-culturing step comprisesco-culturing the second population of antigen-loaded DCs with theenriched population of activated T cells in an initial second co-culturemedium comprising an immune checkpoint inhibitor and optionally one ormore cytokines (e.g., a plurality of cytokines) to provide a secondco-culture; and adding an anti-CD3 antibody (e.g., OKT-3) and optionallyone or more cytokines to the second co-culture to obtain a population oftumor antigen-specific T cells. In some embodiments, the anti-CD3antibody is added to the second co-culture no more than about 3 days(e.g., about 2 days) after the second co-culturing step starts. In someembodiments, the initial second co-culture medium comprises IL-2, IL-7,IL-15 and IL-21 and an anti-PD-1 antibody. In some embodiments, theratio between the population of tumor antigen-specific T cells and thepopulation of antigen-loaded APCs is about 1:1 to about 20:1 (e.g.,about 4:1). In some embodiments, the method further comprises a thirdco-culturing step comprising co-culturing a population of the tumorantigen-specific T cells with a population of APCs (e.g., PBMCs, DCs, orcell line APCs) loaded with one or more target tumor antigen peptidesfrom the plurality of the tumor antigen peptides to obtain a secondpopulation of tumor antigen-specific T cells, wherein the secondpopulation of tumor antigen-specific T cells are subjected tonext-generation sequencing in the sequencing step. In some embodiments,the population of tumor antigen-specific T cells and the population ofantigen-loaded APCs are co-cultured for about 5 to 9 days (e.g., about 7days). In some embodiments, the population of tumor antigen-specific Tcells and the population of antigen-loaded APCs are co-cultured in athird co-culture medium comprising one or more cytokines (such as IL-2or a plurality of cytokines, e.g., IL-2, IL-7, IL-15) and an anti-CD3antibody (e.g., OKT3). In some embodiments, the third co-culturing stepis repeated (e.g., once or twice). In some embodiments, the methodfurther comprises identifying a target tumor epitope from the targettumor antigen peptide, wherein the target tumor epitope elicits specificresponse by the enriched population of activated T cells, and contactinga population of APCs with the target tumor epitope to obtain thepopulation of APCs loaded with the target tumor antigen peptide. In someembodiments, the individual has response (PR), complete response (CR),or stable disease (SD) after receiving the MASCT.

The TCRs can be cloned from any individual who has clinically benefittedfrom a MASCT, including any one or combinations of the MASCT methodsdescribed in the “MASCT” subsection. In some embodiments, the individualhas a partial response (PR) for at least 6 about months (e.g., at leastabout 1 year, 2 years, or more) after receiving the MASCT. In someembodiments, the individual has a complete response (CR) for at least 6about months (e.g., at least about 1 year, 2 years, or more) afterreceiving the MASCT. In some embodiments, the individual has a stabledisease (SD) after receiving the MASCT.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) optionally administering to the individual an effectiveamount of DCs loaded with a plurality of tumor antigen peptidescomprising the target tumor antigen peptide; b) co-culturing apopulation of DCs loaded with the plurality of tumor antigen peptidesand a first population of T cells to obtain a population of activated Tcells; c) administering to the individual an effective amount of theactivated T cells; d) obtaining a second population of T cells from theindividual after achieving PR, CR, or SD after the administration of theactivated T cells; e) a first co-culturing step comprising co-culturinga first population of DCs loaded with the target tumor antigen peptidewith the second population of T cells to obtain a first co-culture; f)an enrichment step comprising subjecting the co-culture to an enrichmentprocess to obtain enriched activated T cells; g) a second co-culturingstep comprising co-culturing the enriched activated T cells with asecond population of DCs loaded with the target tumor antigen peptide toobtain a population of tumor antigen-specific T cells, wherein at leastabout 10% (e.g., at least about 20%, or at least about 50%) of the tumorantigen-specific T cells specifically responds to the target tumorantigen peptide; and h) a sequencing step, comprising subjecting thetumor antigen-specific T cells to next-generation sequencing (e.g.,single-cell sequencing) to identify a plurality of pairs of genesencoding TCRα and TCRβ, thereby providing the plurality of T cellreceptors based on paired genes encoding TCRα and TCRβ. In someembodiments, the first co-culturing step is carried out for about 1 toabout 3 days prior to the enrichment step. In some embodiments, theratio between the population of T cells to the first population ofantigen-loaded DCs is no more than about 30:1 (e.g., about 20:1, 15:1 or10:1). In some embodiments, the second population of T cells in thefirst co-culturing step is present in PBMCs. In some embodiments, thefirst population of antigen-loaded DCs and the second population of Tcells are co-cultured in a first co-culture medium comprising one ormore cytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2,IL-7, IL-15 and IL-21) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody, such as SHR-1210). In some embodiments, theenrichment step comprises contacting the first co-culture with APCs(e.g., DCs or PBMCs) loaded with the plurality of target tumor antigenpeptides to obtain a stimulated co-culture, and isolating from thestimulated co-culture an enriched population of activated T cells usinga ligand that specifically recognizes a cytokine (e.g., IFNy) or a cellsurface molecule. In some embodiments, the ratio between the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs is about 1:1 to about 20:1 (e.g., about 1:1, 2:1 or4:1). In some embodiments, the enriched population of activated T cellsand the second population of antigen-loaded DCs are co-cultured forabout 12 to 25 days. In some embodiments, the second co-culturing stepcomprises co-culturing the second population of antigen-loaded DCs withthe enriched population of activated T cells in an initial secondco-culture medium comprising an immune checkpoint inhibitor andoptionally one or more cytokines (e.g., a plurality of cytokines) toprovide a second co-culture; and adding an anti-CD3 antibody (e.g.,OKT-3) and optionally one or more cytokines to the second co-culture toobtain a population of tumor antigen-specific T cells. In someembodiments, the anti-CD3 antibody is added to the second co-culture nomore than about 3 days (e.g., about 2 days) after the secondco-culturing step starts. In some embodiments, the initial secondco-culture medium comprises IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1antibody. In some embodiments, the ratio between the population of tumorantigen-specific T cells and the population of antigen-loaded APCs isabout 1:1 to about 20:1 (e.g., about 4:1). In some embodiments, themethod further comprises a third co-culturing step comprisingco-culturing a population of the tumor antigen-specific T cells with apopulation of APCs (e.g., PBMCs, DCs, or cell line APCs) loaded with oneor more target tumor antigen peptides from the plurality of the tumorantigen peptides to obtain a second population of tumor antigen-specificT cells, wherein the second population of tumor antigen-specific T cellsare subjected to next-generation sequencing in the sequencing step. Insome embodiments, the population of tumor antigen-specific T cells andthe population of antigen-loaded APCs are co-cultured for about 5 to 9days (e.g., about 7 days). In some embodiments, the population of tumorantigen-specific T cells and the population of antigen-loaded APCs areco-cultured in a third co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, thethird co-culturing step is repeated (e.g., once or twice). In someembodiments, the method further comprises identifying a target tumorepitope from the target tumor antigen peptide, wherein the target tumorepitope elicits specific response by the enriched population ofactivated T cells, and contacting a population of APCs with the targettumor epitope to obtain the population of APCs loaded with the targettumor antigen peptide.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) contacting a population of DCs with a plurality of tumorantigen peptides comprising the target tumor antigen peptide to obtain apopulation of DCs loaded with the plurality of tumor antigen peptides;b) culturing the population of DCs loaded with the plurality of tumorantigen peptides in a DC maturation medium comprising MPLA; c)optionally administering to the individual an effective amount of theDCs loaded with the plurality of tumor antigen peptides; d) co-culturinga population of DCs loaded with the plurality of tumor antigen peptidesand a first population of T cells to obtain a population of activated Tcells; e) administering to the individual an effective amount of theactivated T cells; f) obtaining a second population of T cells from theindividual after achieving PR, CR, or SD after the administration of theactivated T cells; g) a first co-culturing step comprising co-culturinga first population of DCs loaded with the target tumor antigen peptidewith the second population of T cells to obtain a first co-culture; h)an enrichment step comprising subjecting the co-culture to an enrichmentprocess to obtain enriched activated T cells; i) a second co-culturingstep comprising co-culturing the enriched activated T cells with asecond population of DCs loaded with the target tumor antigen peptide toobtain a population of tumor antigen-specific T cells, wherein at leastabout 10% (e.g., at least about 20%, or at least about 50%) of the tumorantigen-specific T cells specifically responds to the target tumorantigen peptide; and j) a sequencing step, comprising subjecting thetumor antigen-specific T cells to next-generation sequencing (e.g.,single-cell sequencing) to identify a plurality of pairs of genesencoding TCRα and TCRβ, thereby providing the plurality of T cellreceptors based on paired genes encoding TCRα and TCRβ. In someembodiments, the DC maturation medium comprises INFy, MPLA and PGE2. Insome embodiments, the first co-culturing step is carried out for about 1to about 3 days prior to the enrichment step. In some embodiments, theratio between the population of T cells to the first population ofantigen-loaded DCs is no more than about 30:1 (e.g., about 20:1, 15:1 or10:1). In some embodiments, the second population of T cells in thefirst co-culturing step is present in PBMCs. In some embodiments, thefirst population of antigen-loaded DCs and the second population of Tcells are co-cultured in a first co-culture medium comprising one ormore cytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2,IL-7, IL-15 and IL-21) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody, such as SHR-1210). In some embodiments, theenrichment step comprises contacting the first co-culture with APCs(e.g., DCs or PBMCs) loaded with the plurality of target tumor antigenpeptides to obtain a stimulated co-culture, and isolating from thestimulated co-culture an enriched population of activated T cells usinga ligand that specifically recognizes a cytokine (e.g., IFNy) or a cellsurface molecule. In some embodiments, the ratio between the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs is about 1:1 to about 20:1 (e.g., about 1:1, 2:1 or4:1). In some embodiments, the enriched population of activated T cellsand the second population of antigen-loaded DCs are co-cultured forabout 12 to 25 days. In some embodiments, the second co-culturing stepcomprises co-culturing the second population of antigen-loaded DCs withthe enriched population of activated T cells in an initial secondco-culture medium comprising an immune checkpoint inhibitor andoptionally one or more cytokines (e.g., a plurality of cytokines) toprovide a second co-culture; and adding an anti-CD3 antibody (e.g.,OKT-3) and optionally one or more cytokines to the second co-culture toobtain a population of tumor antigen-specific T cells. In someembodiments, the anti-CD3 antibody is added to the second co-culture nomore than about 3 days (e.g., about 2 days) after the secondco-culturing step starts. In some embodiments, the initial secondco-culture medium comprises IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1antibody. In some embodiments, the ratio between the population of tumorantigen-specific T cells and the population of antigen-loaded APCs isabout 1:1 to about 20:1 (e.g., about 4:1). In some embodiments, themethod further comprises a third co-culturing step comprisingco-culturing a population of the tumor antigen-specific T cells with apopulation of APCs (e.g., PBMCs, DCs, or cell line APCs) loaded with oneor more target tumor antigen peptides from the plurality of the tumorantigen peptides to obtain a second population of tumor antigen-specificT cells, wherein the second population of tumor antigen-specific T cellsare subjected to next-generation sequencing in the sequencing step. Insome embodiments, the population of tumor antigen-specific T cells andthe population of antigen-loaded APCs are co-cultured for about 5 to 9days (e.g., about 7 days). In some embodiments, the population of tumorantigen-specific T cells and the population of antigen-loaded APCs areco-cultured in a third co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, thethird co-culturing step is repeated (e.g., once or twice). In someembodiments, the method further comprises identifying a target tumorepitope from the target tumor antigen peptide, wherein the target tumorepitope elicits specific response by the enriched population ofactivated T cells, and contacting a population of APCs with the targettumor epitope to obtain the population of APCs loaded with the targettumor antigen peptide.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) optionally administering to the individual an effectiveamount of the DCs loaded with the plurality of tumor antigen peptides;b) co-culturing a population of DCs loaded with the plurality of tumorantigen peptides and a first population of T cells in an initialco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines, e.g., IL-2, IL-7, IL-15 and IL-21) and an immunecheckpoint inhibitor to provide a co-culture; and adding an anti-CD3antibody to the co-culture at about 3 to 7 days (e.g., about 5 days)after the co-culturing starts, thereby obtaining the population ofactivated T cells; c) administering to the individual an effectiveamount of the activated T cells; d) obtaining a second population of Tcells from the individual after achieving PR, CR, or SD after theadministration of the activated T cells; e) a first co-culturing stepcomprising co-culturing a first population of DCs loaded with the targettumor antigen peptide with the second population of T cells to obtain afirst co-culture; f) an enrichment step comprising subjecting theco-culture to an enrichment process to obtain enriched activated Tcells; g) a second co-culturing step comprising co-culturing theenriched activated T cells with a second population of DCs loaded withthe target tumor antigen peptide to obtain a population of tumorantigen-specific T cells, wherein at least about 10% (e.g., at leastabout 20%, or at least about 50%) of the tumor antigen-specific T cellsspecifically responds to the target tumor antigen peptide; and h) asequencing step, comprising subjecting the tumor antigen-specific Tcells to next-generation sequencing (e.g., single-cell sequencing) toidentify a plurality of pairs of genes encoding TCRα and TCRβ, therebyproviding the plurality of T cell receptors based on paired genesencoding TCRα and TCRβ. In some embodiments, the ratio between thepopulation of T cells to the first population of antigen-loaded DCs isno more than about 30:1 (e.g., about 20:1, 15:1 or 10:1). In someembodiments, the second population of T cells in the first co-culturingstep is present in PBMCs. In some embodiments, the first population ofantigen-loaded DCs and the second population of T cells are co-culturedin a first co-culture medium comprising one or more cytokines (such asIL-2 or a plurality of cytokines, e.g., IL-2, IL-7, IL-15 and IL-21) andan immune checkpoint inhibitor (e.g., anti-PD-1 antibody, such asSHR-1210). In some embodiments, the enrichment step comprises contactingthe first co-culture with APCs (e.g., DCs or PBMCs) loaded with theplurality of target tumor antigen peptides to obtain a stimulatedco-culture, and isolating from the stimulated co-culture an enrichedpopulation of activated T cells using a ligand that specificallyrecognizes a cytokine (e.g., IFNy) or a cell surface molecule. In someembodiments, the ratio between the enriched population of activated Tcells and the second population of antigen-loaded DCs is about 1:1 toabout 20:1 (e.g., about 1:1, 2:1 or 4:1). In some embodiments, theenriched population of activated T cells and the second population ofantigen-loaded DCs are co-cultured for about 12 to 25 days. In someembodiments, the second co-culturing step comprises co-culturing thesecond population of antigen-loaded DCs with the enriched population ofactivated T cells in an initial second co-culture medium comprising animmune checkpoint inhibitor and optionally one or more cytokines (e.g.,a plurality of cytokines) to provide a second co-culture; and adding ananti-CD3 antibody (e.g., OKT-3) and optionally one or more cytokines tothe second co-culture to obtain a population of tumor antigen-specific Tcells. In some embodiments, the anti-CD3 antibody is added to the secondco-culture no more than about 3 days (e.g., about 2 days) after thesecond co-culturing step starts. In some embodiments, the initial secondco-culture medium comprises IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1antibody. In some embodiments, the ratio between the population of tumorantigen-specific T cells and the population of antigen-loaded APCs isabout 1:1 to about 20:1 (e.g., about 4:1). In some embodiments, themethod further comprises a third co-culturing step comprisingco-culturing a population of the tumor antigen-specific T cells with apopulation of APCs (e.g., PBMCs, DCs, or cell line APCs) loaded with oneor more target tumor antigen peptides from the plurality of the tumorantigen peptides to obtain a second population of tumor antigen-specificT cells, wherein the second population of tumor antigen-specific T cellsare subjected to next-generation sequencing in the sequencing step. Insome embodiments, the population of tumor antigen-specific T cells andthe population of antigen-loaded APCs are co-cultured for about 5 to 9days (e.g., about 7 days). In some embodiments, the population of tumorantigen-specific T cells and the population of antigen-loaded APCs areco-cultured in a third co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, thethird co-culturing step is repeated (e.g., once or twice). In someembodiments, the method further comprises identifying a target tumorepitope from the target tumor antigen peptide, wherein the target tumorepitope elicits specific response by the enriched population ofactivated T cells, and contacting a population of APCs with the targettumor epitope to obtain the population of APCs loaded with the targettumor antigen peptide.

In some embodiments, there is provided a method of obtaining a pluralityof TCRs specifically recognizing a target tumor antigen peptide,comprising: a) contacting a population of DCs with a plurality of tumorantigen peptides comprising the target tumor antigen peptide to obtain apopulation of DCs loaded with the plurality of tumor antigen peptides;b) culturing the population of DCs loaded with the plurality of tumorantigen peptides in a DC maturation medium comprising MPLA; c)optionally administering to the individual an effective amount of theDCs loaded with the plurality of tumor antigen peptides; d) co-culturinga population of DCs loaded with the plurality of tumor antigen peptidesand a first population of T cells in an initial co-culture mediumcomprising one or more cytokines (such as IL-2 or a plurality ofcytokines) and an immune checkpoint inhibitor to provide a co-culture;and adding an anti-CD3 antibody to the co-culture at about 3 to 7 days(e.g., about 5 days) after the co-culturing starts, thereby obtainingthe population of activated T cells; e) administering to the individualan effective amount of the activated T cells; f) obtaining a secondpopulation of T cells from the individual after achieving PR, CR, or SDafter the administration of the activated T cells; g) a firstco-culturing step comprising co-culturing a first population of DCsloaded with the target tumor antigen peptide with the second populationof T cells to obtain a first co-culture; h) an enrichment stepcomprising subjecting the co-culture to an enrichment process to obtainenriched activated T cells; i) a second co-culturing step comprisingco-culturing the enriched activated T cells with a second population ofDCs loaded with the target tumor antigen peptide to obtain a populationof tumor antigen-specific T cells, wherein at least about 10% (e.g., atleast about 20%, or at least about 50%) of the tumor antigen-specific Tcells specifically responds to the target tumor antigen peptide; and j)a sequencing step, comprising subjecting the tumor antigen-specific Tcells to next-generation sequencing (e.g., single-cell sequencing) toidentify a plurality of pairs of genes encoding TCRα and TCRβ, therebyproviding the plurality of T cell receptors based on paired genesencoding TCRα and TCRβ. In some embodiments, the DC maturation mediumcomprises INFy, MPLA and PGE2. In some embodiments, the firstco-culturing step is carried out for about 1 to about 3 days prior tothe enrichment step. In some embodiments, the ratio between thepopulation of T cells to the first population of antigen-loaded DCs isno more than about 30:1 (e.g., about 20:1, 15:1 or 10:1). In someembodiments, the second population of T cells in the first co-culturingstep is present in PBMCs. In some embodiments, the first population ofantigen-loaded DCs and the second population of T cells are co-culturedin a first co-culture medium comprising one or more cytokines (such asIL-2 or a plurality of cytokines, e.g., IL-2, IL-7, IL-15 and IL-21) andan immune checkpoint inhibitor (e.g., anti-PD-1 antibody, such asSHR-1210). In some embodiments, the enrichment step comprises contactingthe first co-culture with APCs (e.g., DCs or PBMCs) loaded with theplurality of target tumor antigen peptides to obtain a stimulatedco-culture, and isolating from the stimulated co-culture an enrichedpopulation of activated T cells using a ligand that specificallyrecognizes a cytokine (e.g., IFNy) or a cell surface molecule. In someembodiments, the ratio between the enriched population of activated Tcells and the second population of antigen-loaded DCs is about 1:1 toabout 20:1 (e.g., about 1:1, 2:1 or 4:1). In some embodiments, theenriched population of activated T cells and the second population ofantigen-loaded DCs are co-cultured for about 12 to 25 days. In someembodiments, the second co-culturing step comprises co-culturing thesecond population of antigen-loaded DCs with the enriched population ofactivated T cells in an initial second co-culture medium comprising oneor more cytokines (such as IL-2 or a plurality of cytokines) and animmune checkpoint inhibitor to provide a second co-culture; and addingan anti-CD3 antibody (e.g., OKT-3) to the second co-culture to obtain apopulation of tumor antigen-specific T cells. In some embodiments, theanti-CD3 antibody is added to the second co-culture no more than about 3days (e.g., about 2 days) after the second co-culturing step starts. Insome embodiments, the initial second co-culture medium comprises IL-2,IL-7, IL-15 and IL-21 and an anti-PD-1 antibody. In some embodiments,the ratio between the population of tumor antigen-specific T cells andthe population of antigen-loaded APCs is about 1:1 to about 20:1 (e.g.,about 4:1). In some embodiments, the method further comprises a thirdco-culturing step comprising co-culturing a population of the tumorantigen-specific T cells with a population of APCs (e.g., PBMCs, DCs, orcell line APCs) loaded with one or more target tumor antigen peptidesfrom the plurality of the tumor antigen peptides to obtain a secondpopulation of tumor antigen-specific T cells, wherein the secondpopulation of tumor antigen-specific T cells are subjected tonext-generation sequencing in the sequencing step. In some embodiments,the population of tumor antigen-specific T cells and the population ofantigen-loaded APCs are co-cultured for about 5 to 9 days (e.g., about 7days). In some embodiments, the population of tumor antigen-specific Tcells and the population of antigen-loaded APCs are co-cultured in athird co-culture medium comprising one or more cytokines (such as IL-2or a plurality of cytokines, e.g., IL-2, IL-7, IL-15) and an anti-CD3antibody (e.g., OKT3). In some embodiments, the third co-culturing stepis repeated (e.g., once or twice). In some embodiments, the methodfurther comprises identifying a target tumor epitope from the targettumor antigen peptide, wherein the target tumor epitope elicits specificresponse by the enriched population of activated T cells, and contactinga population of APCs with the target tumor epitope to obtain thepopulation of APCs loaded with the target tumor antigen peptide.

In some embodiments, the TCR is cloned from an individual that respondsto the MASCT method, for example, an individual having reduced CTCnumber or a low CTC number after the MASCT, an individual having aclinical evaluation of Stable Disease (SD), Complete Response (CR), orPartial Response (PR). In some embodiments, the individual remains PR,CR or SD for at least about 6 months (e.g., at least about 1 year, 2years, or more). In some embodiments, the individual has a strongspecific immune response against the target tumor antigen peptide.Specific immune response against a target tumor antigen peptide may bedetermined using any known methods in the art, for example, by measuringlevels of cytotoxic factor (such as perforin or granzyme B), or cytokinerelease (such as IFNy or TNFα) from T cells (or PBMCs) after stimulationby the target tumor antigen peptide. An antibody-based assay, such asELISPOT, may be used to quantify the cytotoxic factor, or cytokine (suchas IFNy) levels. In some embodiments, the cytokine (such as IFNy)release level from T cells (or PBMCs) in response to a target tumorantigen peptide is normalized to a reference, such as a baselinecytokine release level, or a nonspecific cytokine release level of fromT cells (or PBMCs) in response to an irrelevant peptide, to provide acytokine (such as IFNy) fold change value. In some embodiments, acytokine (such as IFNy) fold change value of more than about any of 1.2,1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, or more in an ELISPOT assay indicatestrong specific immune response against the target tumor antigenpeptide. In some embodiments, the method of cloning a TCR furthercomprises determining the specific immune response of each of theplurality of tumor antigen peptides in the individual, such as in a PBMCsample of the individual.

The T cell may be isolated from a biological sample from the individualafter receiving the MASCT. In some embodiments, the biological sample isobtained from the individual after one cycle of MASCT. In someembodiments, the biological sample is obtained from the individual afterat least any of 2, 3, 4, 5, or more cycles of MASCT. In someembodiments, the biological sample is obtained from the individual afterat least about any of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 2 months, or 3 months after receiving the MASCT. In someembodiments, the biological sample is obtained from the individual afterno more than about any of 6 months, 3 months, 2 months, 1 month, or lessafter receiving the MASCT. In some embodiments, the biological sample isa blood sample. In some embodiments, the biological sample is a PBMCsample. In some embodiments, the biological sample is a T cell sample.In some embodiments, the biological sample is a tumor sample containingCTLs. T cells may be isolated from the biological sample using any knownmethods in the art, for example, by flow cytometry or centrifugationmethods. In some embodiments, a plurality of T cells obtained from thebiological sample are screened for their specific immune responseagainst the plurality of tumor antigen peptides, for example, bystaining with multimers (such as pentamers or dextramers), or bydetermining the level of cytotoxic factor (such as perforin or granzymeB), or cytokine release (such as IFNy or TNFα) by the cell.

The target tumor antigen peptide that the T cell specifically recognizescan be any tumor antigen peptide or fragment thereof from the tumorantigen peptide pool(s) used for the MASCT. In some embodiments, thetarget tumor antigen peptide comprises an MHC-I restricted epitope. Insome embodiments, the target tumor antigen peptide comprises an MHC-IIrestricted epitope. In some embodiments, the target tumor antigenpeptide is a general cancer tumor antigen peptide. In some embodiments,the target tumor antigen peptide is a cancer-type specific tumor antigenpeptide. In some embodiments, the target tumor antigen peptide is aneoantigen peptide. In some embodiments, the target tumor antigenpeptide is derived from a tumor antigen selected from the groupconsisting of hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1, RGS5, MMP7,VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3, CDCA1, WT1, KRAS, PARP4,MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7, HPV58-E6, HPV58-E7,HBcAg, HBV polymerase, GPC3, SSX, and AFP. In some embodiments, thetarget tumor antigen peptide comprises an epitope derived from CEA, RGS5or HPV18-E7.

Any known next-generation sequencing methods can be used to sequence thetumor antigen-specific T cells to provide a plurality of pairs of genesencoding TCRα and TCRβ. In some embodiments, the next-generationsequencing is single-cell next generation sequencing. In someembodiments, the next-generation sequencing is bulk next-generationsequencing. In some embodiments, the sequencing step comprisesnext-generation sequencing of immune repertoire of TCRs of the tumorantigen-specific T cells. In some embodiments, the sequencing stepcomprises amplification of genes encoding TCRα and TCRβ from the tumorantigen-specific T cells (e.g., single cells or a population of cells)to provide a sample of amplified nucleic acids, and subjecting theamplified nucleic acids to next-generation sequencing. In someembodiments, the genes encoding TCRα and TCRβ are amplified using PCRmethods with primers that specifically annealing to known TCR variabledomains. In some embodiments, amplicon rescued multiplex PCR (orarm-PCR) is used to amplify the genes encoding TCRα and TCRβ. See, forexample, U.S. Pat. No. 7,999,092. In some embodiments, the tumorantigen-specific T cells are subjected to both bulk next-generationsequencing data and single-cell next generation sequencing to identify aplurality of pairs of genes encoding TCRα and TCRβ. In some embodiments,the sequencing step comprises bulk sequencing of a first portion of thetumor antigen-specific T cells to provide a plurality of genes encodingTCRα and TCRβ, and single-cell sequencing of a second portion of thetumor antigen-specific T cells providing cognate pairing information ofthe plurality of genes encoding TCRα and TCRβ, thereby providing aplurality of TCRs based on paired genes encoding TCRα and TCRβ.

Commercial kits and services are available for single-cell nextgeneration sequencing of TCRs, including, but not limited to IPAR®(iReportoire), IMMUNOSEQ™ (Adaptive Biotech), SMARTER® human TCRa/bprofiling kit (Clontech), ION AMPLISEQ® Immune Repertoire and Assay Plus(Thermofisher). For example, the IPAR® method including two steps of PCRamplification followed by next-generation sequencing. Briefly, a sampleof tumor antigen-specific T cells is subject to single cell plating inone or more 96-well plates. In the first PCR step, RT-PCR is performedin each of the wells of the 96 well-plates with nested, multiplexprimers covering both the alpha and beta locus of TCR with communalforward and reverse binding sites included on the 5′-end of the insideprimers. Included on the C-region gene primer is an in-line barcode,which serves as a plate identifier so that multiple 96-well plates maybe multiplexed on a sequencing flow cell. After the RT-PCR, the productsare rescued. A second PCR is performed with dual-indexed primers thatcomplete the adaptors introduced during the first PCR and provide platepositional information. In this step, each well, and thus each singlecell, is uniquely barcoded. In some cases, bulknext-generation_sequencing of each chain is also performed on RNA fromremaining cells (those cells not subjected to single cell plating).Sequencing data is analyzed using the IPAR® Analyzer software, whichalso facilitates easy comparisons of single-cell sequencing data to thebulk sequencing data. Cognate pairing information of genes encoding TCRαand TCRβ is obtained based on the data in each well as determined by theIPAR® Analyzer. Also, see, for example, iPair Analyzer User’s Guide,iRepertoire Inc.(docs.wixstatic.com/ugd/c9f231_3c322d131c084e908eea039c42304ff7.pdf),the contents of which are incorporated herein by reference.

In some embodiments, the tumor antigen-specific T cells are stimulatedwith APCs (e.g., PBMCs, DCs, or cell line APCs) loaded with the targettumor antigen peptide (i.e., stimulated tumor antigen-specific T cells)prior to the next-generation sequencing. In some embodiments, the tumorantigen-specific T cells stimulated with the APCs loaded with the targettumor antigen peptide are sorted to isolate IFNγ⁺ T cells for thenext-generation sequencing. In some embodiments, a control population ofT cells are subject to the next-generation sequencing to provide abaseline TCRα and TCRβ clonotype profile. In some embodiments, thecontrol population of T cells are unstimulated PBMCs from theindividual. In some embodiments, the control population of T cells aretumor antigen-specific T cells that are stimulated with one or moreirrelevant peptides. In some embodiments, TCRα and TCRβ genes that areonly identified in the sequencing data of stimulated tumorantigen-specific T cells, but not in the sequencing data of the controlpopulation of T cells are selected to provide tumor-specific TCRs. Insome embodiments, TCRα and TCRβ genes that are present at a frequency atleast about any one of 2x, 5x, 10x, 20x, 50x, 100x, 1000x or more in thesequencing data of stimulated tumor antigen-specific T cells than in thesequencing data of the control population of T cells are selected toprovide tumor-specific TCRs.

In some embodiments, a plurality of population of T cells (e.g., PBMCs)from the individual is obtained at different time after the MASCTtreatment. The TCRα and TCRβ genes consistently identified from thetumor antigen-specific T cells prepared using each population of T cellsare selected to provide tumor-specific TCRs. In some embodiments, if aTCRα and a TCRβ genes are found in the sequencing data of two or more(such as 2, 3, 4, 5, 6 or more) populations of tumor antigen-specific Tcells prepared using different samples of T cells from the individual,and the TCRα and TCRβ genes are paired by single-cell sequencing, thenthe pair of TCRα and TCRβ genes is selected to provide a tumor-specificTCR.

Any one of the methods described herein may comprise one or more of thefollowings steps: (i) expressing each pair of genes encoding TCRα andTCRβ in a host immune cell to provide an engineered immune cellexpressing a TCR, and assessing response of the engineered immune cellto the target tumor antigen peptide; (ii) determining the antigenepitope recognized by each TCR; (iii) determining HLA restriction(including MHC I or MHC II restriction, and optionally HLA haplotyperestriction) of each TCR; (iv) determining the avidity andcross-reactivity of each TCR; (v) affinity maturation of each TCR; (vi)enhancing the paring of the TCRα and TCRβ chains in each TCR; and (vii)enhancing the expression of each TCR. An overview of an exemplary methodof obtaining a plurality of TCRs specifically recognizing a target tumorantigen peptide from an individual who has clinically benefitted from aMASCT is shown in FIG. 1 .

In some embodiments, isolated nucleic acids comprising each pair of TCRαand TCRβ genes are synthesized. In some embodiments, for each pair ofTCRα and TCRβ genes, a first isolated nucleic acid encoding the TCRαgene and a second isolated nucleic acid encoding the TCRβ gene aresynthesized. In some embodiments, the TCRα and TCRβ genes are codonoptimized (e.g., for expression in human cells). In some embodiments,each of the TCRα and TCRβ genes is operably linked to a promoter. Insome embodiments, the TCRα and TCRβ genes are operably linked to thesame promoter. In some embodiments, the TCRα and TCRβ genes are operablylinked to different promoters. In some embodiments, the isolated nucleicacids are incorporated in a vector, such as a viral vector, for example,a lentiviral vector.

In some embodiments, the isolated nucleic acids is transduced (such asby a viral vector, or by physical or chemical methods) into a hostimmune cell (such as T cell) to express the TCR encoded by the TCRα andTCRβ genes. In some embodiments, the host immune cell is a CD3⁺ cell. Insome embodiments, the host immune cell is a T cell. In some embodiments,the host immune cell is selected from the group consisting of a PBMC, acytotoxic T cell, a helper T cell, a natural killer T cell, and aregulatory T cell. In some embodiments, the host immune cell expressingthe TCR is assayed for specific immune response to the target tumorantigen peptide for validation. In some embodiments, the host immunecell is derived from a cell line. In some embodiments, the host immunecell is a primary cell. In some embodiments, the host immune cell isderived from a cancer patient. In some embodiments, the host immune cellis derived from a healthy donor.

Further provided is a method of obtaining a TCR specifically recognizinga target tumor antigen peptide using any one of the method of obtaininga plurality of TCRs specifically recognizing a target tumor antigenpeptide as described herein, wherein the TCR is selected based on theresponse of an engineered immune cell expressing the TCR to the targettumor antigen peptide.

HLA restriction of the TCRs may be determined using any known methods inthe art. See, for example, Larche M. Methods Mol. Med. (2008),138:57-72. In some embodiments, the TCR is MHC class I restricted. Insome embodiments, the TCR is MHC class II restricted. In someembodiments, In some embodiments, the tumor-specific TCR has a HLAhaplotype restriction that is predominant in certain a racial groups,including, but not limited to, Africans, African Americans, Asians,Caucasians, Europeans, Hispanics, Pacific Islanders, etc.. In someembodiments, the TCR has a HLA haplotype restriction that is predominantin Asians, for example, HLA-A*1101 or HLA-A*2402-restricted TCR. In someembodiments, the TCR has a HLA haplotype restriction that is predominantin Caucasians, e.g., HLA-DPB1*0401 or HLA-A*0201-restricted TCR.

The TCRs obtained herein may be further engineered to improve thephysical/chemical properties and/or functions of the TCRs. For example,the engineered tumor-specific TCRs may have enhanced expression level,improved stability, enhanced binding affinity to the MHC-targettumor-specific antigen peptide complexes, and/or enhanced signaling. Insome embodiments, the TCRs are engineered based on the MHC subtype ofthe individual receiving immunotherapy treatment using the TCRs. In someembodiments, the engineering comprises mutating one or more positions inthe variable regions of a TCR. In some embodiments, the engineeringcomprises providing a fusion protein comprising one or more domains orfragments of the TCR. In some embodiments, the TCRα and TCRβ chains of aTCR may be engineered to have enhanced pairing. Any known methods in theart for epitope determining, affinity maturation, avidity andcross-reactivity determination, expression enhancement, and pairingenhancement may be used.

It is intended that any of the steps and parameters described herein forpreparing the antigen-loaded APCs, the first, second and thirdco-culturing steps, the enrichment step, the sequencing step, the MASCT,etc., can be combined with each other as if each and every combinationis individually described.

Methods for Preparing Tumor Antigen-Specific T Cells

The present application provides methods of preparing a population of Tcells for TCR cloning, comprising: co-culturing a population of enrichedactivated T cells or a population of stock tumor antigen-specific Tcells with a population of antigen-presenting cells (APCs) loaded withone or more target tumor antigen peptides (referred herein as“antigen-loaded APCs”), wherein the population of enriched activated Tcells or the population of stock tumor antigen-specific T cells isobtained from an individual who has clinically benefitted from a MASCT,and wherein at least about 10% (e.g., at least 20% or 50%) of thepopulation of T cells for TCR cloning specifically responds to thetarget tumor antigen peptide. In some embodiments, the tumor-antigenspecific T cells are obtained using any one of the methods described inInternational Patent Application No. PCT/CN2018/082945, which isincorporated herein in reference by its entirety.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: co-culturing anenriched population of activated T cells with a population of DCs loadedwith a target tumor antigen peptide, wherein the enriched population ofactivated T cells is prepared by subjecting a first co-culture to anenrichment process, and wherein the first co-culture comprises apopulation of T cells and a first population of DCs loaded with thetumor antigen peptide, wherein the population of T cells in the firstco-culture is obtained from an individual who has clinically benefittedfrom a MASCT, and wherein at least about 10% (e.g., at least 20% or 50%)of the population of T cells for TCR cloning specifically responds tothe target tumor antigen peptide. In some embodiments, the ratio betweenthe enriched population of activated T cells and the population ofantigen-loaded DCs is about 1:1 to about 20:1 (e.g., about 1:1 or about2:1). In some embodiments, the enriched population of activated T cellsand the population of antigen-loaded DCs are co-cultured for about 12 to25 days. In some embodiments, the method comprises co-culturing thepopulation of antigen-loaded DCs with the enriched population ofactivated T cells in an initial co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines) and an immunecheckpoint inhibitor (e.g., anti-PD-1 antibody) to provide a co-culture;and adding an anti-CD3 antibody (e.g., OKT-3) and optionally one or morecytokines to the co-culture to obtain a population of tumorantigen-specific T cells. In some embodiments, the anti-CD3 antibody isadded to the co-culture no more than about 3 days (e.g., about 2 days)after the co-culturing step starts.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: a) subjecting a firstco-culture to an enrichment process to obtain an enriched population ofactivated T cells, wherein the first co-culture comprises a firstpopulation of DCs loaded with a target tumor antigen peptide and apopulation of T cells; and b) co-culturing the enriched population ofactivated T cells with a second population of DCs loaded with the targettumor antigen peptide to obtain a population of T cells for TCR cloning,wherein the population of T cells in step a) is obtained from anindividual who has clinically benefitted from a MASCT, and wherein atleast about 10% (e.g., at least 20% or 50%) of the population of T cellsfor TCR cloning specifically responds to the target tumor antigenpeptide. In some embodiments, the enrichment process comprisescontacting the first co-culture with APCs (e.g., PBMCs) loaded with thetarget tumor antigen peptide to obtain a stimulated co-culture, andisolating an enriched population of activated T cells from thestimulated co-culture using a ligand that specifically recognizes acytokine (such as IFNy) or a cell surface molecule. In some embodiments,the ratio between the enriched population of activated T cells and thesecond population of antigen-loaded DCs is about 1:1 to about 20:1(e.g., about 1:1 or about 2:1). In some embodiments, the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs are co-cultured for about 12 to 25 days. In someembodiments, the method comprises co-culturing the second population ofantigen-loaded DCs with the population of T cells in an initial secondco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody) to provide a second co-culture; and adding ananti-CD3 antibody (e.g., OKT3) to the second co-culture to obtain apopulation of tumor antigen-specific T cells. In some embodiments, theanti-CD3 antibody is added to the second co-culture no more than about 3days (e.g., about 2 days) after the second co-culturing step starts.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: a) a firstco-culturing step, comprising co-culturing a first population of DCsloaded with a target tumor antigen peptide with a population of T cellsto obtain a first co-culture comprising activated T cells; b) anenrichment step, comprising subjecting the first co-culture to anenrichment process to obtain an enriched population of activated Tcells; and c) a second co-culturing step, comprising co-culturing theenriched population of activated T cells with a second population of DCsloaded with the target tumor antigen peptide to obtain a population of Tcells for TCR cloning, wherein the population of T cells in the firstco-culturing step is obtained from an individual who has clinicallybenefitted from a MASCT, and wherein at least about 10% (e.g., at least20% or 50%) of the population of T cells for TCR cloning specificallyresponds to the target tumor antigen peptide. In some embodiments, thefirst co-culturing step is carried out for no more than about 7 days(such as about 1-3 days, e.g., about 3 days) prior to the enrichmentstep. In some embodiments, the ratio between the population of T cellsto the first population of antigen-loaded DCs is no more than about 30:1(e.g., about 20:1, 15:1 or 10:1). In some embodiments, the firstpopulation of antigen-loaded DCs and the population of T cells areco-cultured in a first co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15 and IL-21) and an immune checkpoint inhibitor (e.g., anti-PD-1antibody). In some embodiments, the ratio between the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs is about 1:1 to about 20:1 (e.g., about 1:1 or about2:1). In some embodiments, the enriched population of activated T cellsand the second population of antigen-loaded DCs are co-cultured forabout 12 to 25 days. In some embodiments, the method comprisesco-culturing the second population of antigen-loaded DCs with thepopulation of T cells in an initial second co-culture medium comprisingone or more cytokines (such as IL-2 or a plurality of cytokines) and animmune checkpoint inhibitor (e.g., anti-PD-1 antibody) to provide asecond co-culture; and adding an anti-CD3 antibody (e.g., OKT3) to thesecond co-culture to obtain a population of tumor antigen-specific Tcells. In some embodiments, the anti-CD3 antibody is added to the secondco-culture no more than about 3 days (e.g., about 2 days) after thesecond co-culturing step starts.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: a) a firstco-culturing step, comprising co-culturing a first population of DCsloaded with a target tumor antigen peptide with a population of T cellsto obtain a first co-culture comprising activated T cells; b) anenrichment step, comprising contacting the first co-culture with APCs(e.g., PBMCs) loaded with the target tumor antigen peptide to obtain astimulated co-culture, and isolating an enriched population of activatedT cells from the stimulated co-culture using a ligand that specificallyrecognizes a cytokine (such as IFNy) or a cell surface molecule; and c)a second co-culturing step, comprising co-culturing the enrichedpopulation of activated T cells with a second population of DCs loadedwith the target tumor antigen peptide to obtain a population of T cellsfor TCR cloning, wherein the population of T cells in the firstco-culturing step is obtained from an individual who has clinicallybenefitted from a MASCT, and wherein at least about 10% (e.g., at least20% or 50%) of the population of T cells for TCR cloning specificallyresponds to the target tumor antigen peptide. In some embodiments, thefirst co-culturing step is carried out for no more than about 7 days(such as about 1-3 days, e.g., about 3 days) prior to the enrichmentstep. In some embodiments, the ratio between the population of T cellsto the first population of antigen-loaded DCs is no more than about 30:1(e.g., about 20:1, 15:1 or 10:1). In some embodiments, the firstpopulation of antigen-loaded DCs and the population of T cells areco-cultured in a first co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15 and IL-21) and an immune checkpoint inhibitor (e.g., anti-PD-1antibody). In some embodiments, the ratio between the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs is about 1:1 to about 20:1 (e.g., about 1:1 or about2:1). In some embodiments, the enriched population of activated T cellsand the second population of antigen-loaded DCs are co-cultured forabout 12 to 25 days. In some embodiments, the method comprisesco-culturing the second population of antigen-loaded DCs with thepopulation of T cells in an initial second co-culture medium comprisingone or more cytokines (such as IL-2 or a plurality of cytokines) and animmune checkpoint inhibitor (e.g., anti-PD-1 antibody) to provide asecond co-culture; and adding an anti-CD3 antibody (e.g., OKT3) to thesecond co-culture to obtain a population of tumor antigen-specific Tcells. In some embodiments, the anti-CD3 antibody is added to the secondco-culture no more than about 3 days (e.g., about 2 days) after thesecond co-culturing step starts. In some embodiments, the population ofT cells in the first co-culturing step is present in a population ofPBMCs.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: a) a firstco-culturing step, comprising co-culturing a first population of DCsloaded with a target tumor antigen peptide with a population of T cellsto obtain a first co-culture comprising activated T cells; b) anenrichment step, comprising subjecting the first co-culture to anenrichment process to obtain an enriched population of activated Tcells; and c) a second co-culturing step, comprising co-culturing theenriched population of activated T cells with a second population of DCsloaded with the target tumor antigen peptide in an initial secondco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody) to obtain a second co-culture, and adding ananti-CD3 antibody (e.g., OKT3) to the second co-culture to obtain apopulation of T cells for TCR cloning, wherein the population of T cellsin the first co-culturing step is obtained from an individual who hasclinically benefitted from a MASCT, and wherein at least about 10%(e.g., at least 20% or 50%) of the population of T cells for TCR cloningspecifically responds to the target tumor antigen peptide. In someembodiments, the anti-CD3 antibody is added to the second co-culture nomore than about 3 days (e.g., about 2 days) after the secondco-culturing step starts. In some embodiments, the first co-culturingstep is carried out for no more than about 7 days (such as about 1-3days, e.g., about 3 days) prior to the enrichment step. In someembodiments, the ratio between the population of T cells to the firstpopulation of antigen-loaded DCs is no more than about 30:1 (e.g., about20:1, 15:1 or 10:1). In some embodiments, the first population ofantigen-loaded DCs and the population of T cells are co-cultured in afirst co-culture medium comprising one or more cytokines (such as IL-2or a plurality of cytokines, e.g., IL-2, IL-7, IL-15 and IL-21) and animmune checkpoint inhibitor (e.g., anti-PD-1 antibody). In someembodiments, the enrichment process comprises contacting the firstco-culture with APCs (e.g., PBMCs) loaded with the target tumor antigenpeptide to obtain a stimulated co-culture, and isolating an enrichedpopulation of activated T cells from the stimulated co-culture using aligand that specifically recognizes a cytokine (such as IFNy) or a cellsurface molecule. In some embodiments, the ratio between the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs is about 1:1 to about 20:1 (e.g., about 1:1 or about2:1). In some embodiments, the enriched population of activated T cellsand the second population of antigen-loaded DCs are co-cultured forabout 12 to 25 days. In some embodiments, the population of T cells inthe first co-culturing step is present in a population of PBMCs.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: a) contacting a firstpopulation of DCs with a target tumor antigen peptide to obtain a firstpopulation of DCs loaded with the target tumor antigen peptide; b) afirst co-culturing step, comprising co-culturing the first population ofDCs loaded with the target tumor antigen peptide with a population of Tcells to obtain a first co-culture comprising activated T cells; c) anenrichment step, comprising subjecting the first co-culture to anenrichment process to obtain an enriched population of activated Tcells; d) contacting a second population of dendritic cells with thetarget tumor antigen peptide to obtain a second population of DCs loadedwith the target tumor antigen peptide; and e) a second co-culturingstep, comprising co-culturing the enriched population of activated Tcells with the second population of DCs loaded with the target tumorantigen peptide in an initial second co-culture medium comprising one ormore cytokines (such as IL-2 or a plurality of cytokines) and an immunecheckpoint inhibitor (e.g., anti-PD-1 antibody) to obtain a secondco-culture, and adding an anti-CD3 antibody (e.g., OKT3) to the secondco-culture to obtain a population of T cells for TCR cloning, whereinthe population of T cells in the first co-culturing step is obtainedfrom an individual who has clinically benefitted from a MASCT, andwherein at least about 10% (e.g., at least 20% or 50%) of the populationof T cells for TCR cloning specifically responds to the target tumorantigen peptide. In some embodiments, the anti-CD3 antibody is added tothe second co-culture no more than about 3 days (e.g., about 2 days)after the second co-culturing step starts. In some embodiments, thefirst co-culturing step is carried out for no more than about 7 days(such as about 1-3 days, e.g., about 3 days) prior to the enrichmentstep. In some embodiments, the ratio between the population of T cellsto the first population of antigen-loaded DCs is no more than about 30:1(e.g., about 20:1, 15:1 or 10:1). In some embodiments, the firstpopulation of antigen-loaded DCs and the population of T cells areco-cultured in a first co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15 and IL-21) and an immune checkpoint inhibitor (e.g., anti-PD-1antibody). In some embodiments, the enrichment process comprisescontacting the first co-culture with APCs (e.g., PBMCs) loaded with thetarget tumor antigen peptide to obtain a stimulated co-culture, andisolating an enriched population of activated T cells from thestimulated co-culture using a ligand that specifically recognizes acytokine (such as IFNy) or a cell surface molecule. In some embodiments,the ratio between the enriched population of activated T cells and thesecond population of antigen-loaded DCs is about 1:1 to about 20:1(e.g., about 1:1 or about 2:1). In some embodiments, the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs are co-cultured for about 12 to 25 days. In someembodiments, the population of T cells in the first co-culturing step ispresent in a population of PBMCs.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising a) contacting a firstpopulation of DCs with a target tumor antigen peptide to obtain a firstpopulation of DCs loaded with the target tumor antigen peptide; b)culturing the first population of DCs loaded with the target tumorantigen peptide in a DC maturation medium comprising a toll-likereceptor (TLR) agonist; c) a first co-culturing step, comprisingco-culturing the first population of DCs loaded with the target tumorantigen peptide with the population of T cells in a first co-culturemedium comprising one or more cytokines (such as IL-2 or a plurality ofcytokines, e.g., IL-2, IL-7, IL-15 and IL-21), an immune checkpointinhibitor (e.g., anti-PD-1 antibody) to obtain a first co-culturecomprising activated T cells; d) an enrichment step, comprisingcontacting the first co-culture with PBMCs loaded with the target tumorantigen peptide to obtain a stimulated co-culture, and isolating anenriched population of activated T cells from the stimulated co-cultureusing a ligand that specifically recognizes a cytokine (such as IFNy) ora cell surface molecule; e) contacting a second population of DCs withthe target tumor antigen peptide to obtain a second population ofantigen-loaded DCs; f) culturing the second population of antigen-loadedDCs in a DC maturation medium comprising a toll-like receptor (TLR)agonist; and g) a second co-culturing step, comprising co-culturing theenriched population of activated T cells with the second population ofDCs loaded with the target tumor antigen peptide in a second initialco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody) to obtain a second co-culture, and adding ananti-CD3 antibody (e.g., OKT3) to the second co-culture to provide apopulation of T cells for TCR cloning, wherein the population of T cellsin the first co-culturing step is obtained from an individual who hasclinically benefitted from a MASCT, and wherein at least about 10%(e.g., at least 20% or 50%) of the population of T cells for TCR cloningspecifically responds to the target tumor antigen peptide. In someembodiments, the anti-CD3 antibody is added to the second co-culture nomore than about 3 days (e.g., about 2 days) after the secondco-culturing step starts. In some embodiments, the first co-culturingstep is carried out for no more than about 7 days (such as about 1-3days, e.g., about 3 days) prior to the enrichment step. In someembodiments, the ratio between the population of T cells to the firstpopulation of antigen-loaded DCs is no more than about 30:1 (e.g., about20:1, 15:1 or 10:1). In some embodiments, the first population ofantigen-loaded DCs and the population of T cells are co-cultured in afirst co-culture medium comprising one or more cytokines (such as IL-2or a plurality of cytokines, e.g., IL-2, IL-7, IL-15 and IL-21) and animmune checkpoint inhibitor (e.g., anti-PD-1 antibody). In someembodiments, the ratio between the enriched population of activated Tcells and the second population of antigen-loaded DCs is about 1:1 toabout 20:1 (e.g., about 1:1 or about 2:1). In some embodiments, theenriched population of activated T cells and the second population ofantigen-loaded DCs are co-cultured for about 12 to 25 days. In someembodiments, the population of T cells in the first co-culturing step ispresent in a population of PBMCs. In some embodiments, the DC maturationmedium comprises INFy, MPLA and PGE2. In some embodiments, the firstpopulation of DCs and/or the second population of DCs is obtained byinducing differentiation of a population of monocytes from PBMCs.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: co-culturing apopulation of tumor antigen-specific T cells with a population of APCs(e.g., PBMCs, DCs, or cell line APCs) loaded with one or more targettumor antigen peptides. In some embodiments, the population of tumorantigen-specific T cells is obtained using any one of the methods ofpreparing T cells for cloning TCR described above. In some embodiments,the population of tumor antigen-specific T cells is obtained from thePBMCs of an individual that has clinically benefitted from a MASCT. Insome embodiments, the ratio between the population of tumorantigen-specific T cells and the population of antigen-loaded APCs isabout 1:1 to about 20:1 (e.g., about 1:1, 1:2 or 1:4). In someembodiments, the population of tumor antigen-specific T cells and thepopulation of antigen-loaded APCs are co-cultured for about 5 to 9(e.g., about 7) days. In some embodiments, the population of tumorantigen-specific T cells and the population of antigen-loaded APCs areco-cultured in a third co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, theco-culturing is repeated, e.g., once or twice. In some embodiments, thepopulation of the tumor antigen-specific T cells is obtained from afrozen stock of the tumor antigen-specific T cells.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: a) a firstco-culturing step, comprising co-culturing a first population of DCsloaded with a target tumor antigen peptide with a population of T cellsto obtain a first co-culture comprising activated T cells; b) anenrichment step, comprising subjecting the first co-culture to anenrichment process to obtain an enriched population of activated Tcells; c) a second co-culturing step, comprising co-culturing theenriched population of activated T cells with a second population of DCsloaded with the target tumor antigen peptide to obtain a firstpopulation of tumor antigen-specific T cells; d) a third co-culturingstep, comprising co-culturing a subpopulation of tumor antigen-specificT cells from the first population of tumor antigen-specific T cells witha third population of APCs (e.g., DCs, PBMCs, or cell line APCs such asLCL) loaded with the target tumor antigen peptide, thereby providing apopulation of T cells for TCR cloning, wherein the population of T cellsin the first co-culturing step is obtained from an individual who hasclinically benefitted from a MASCT, and wherein at least about 10%(e.g., at least 20% or 50%) of the population of T cells for TCR cloningspecifically responds to the target tumor antigen peptide. In someembodiments, the ratio between the subpopulation of tumorantigen-specific T cells and the third population of antigen-loaded DCsis about 1:1 to about 20:1 (e.g., about 1:1, 1:2 or 1:4). In someembodiments, the subpopulation of tumor antigen-specific T cells and thethird population of antigen-loaded DCs are co-cultured for about 5 to 9(e.g., about 7) days. In some embodiments, the subpopulation of tumorantigen-specific T cells and the third population of antigen-loaded DCsare co-cultured in a third co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines, e.g., IL-2, IL-7,IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, thethird co-culturing step is repeated, e.g., once, twice or three times.In some embodiments, the subpopulation of tumor antigen-specific T cellsis obtained from a frozen stock of the first population of tumorantigen-specific T cells. In some embodiments, the first co-culturingstep is carried out for no more than about 7 days (such as about 1-3days, e.g., about 3 days) prior to the enrichment step. In someembodiments, the ratio between the population of T cells to the firstpopulation of antigen-loaded DCs is no more than about 30:1 (e.g., about20:1, 15:1 or 10:1). In some embodiments, the first population ofantigen-loaded DCs and the population of T cells are co-cultured in afirst co-culture medium comprising one or more cytokines (such as IL-2or a plurality of cytokines, e.g., IL-2, IL-7, IL-15 and IL-21) and animmune checkpoint inhibitor (e.g., anti-PD-1 antibody). In someembodiments, the ratio between the enriched population of activated Tcells and the second population of antigen-loaded DCs is about 1:1 toabout 20:1 (e.g., about 1:1 or about 2:1). In some embodiments, theenriched population of activated T cells and the second population ofantigen-loaded DCs are co-cultured for about 12 to 25 days. In someembodiments, the method comprises co-culturing the second population ofantigen-loaded DCs with the population of T cells in an initial secondco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody) to provide a second co-culture; and adding ananti-CD3 antibody (e.g., OKT3) to the second co-culture to obtain apopulation of tumor antigen-specific T cells. In some embodiments, theanti-CD3 antibody is added to the second co-culture no more than about 3days (e.g., about 2 days) after the second co-culturing step starts. Insome embodiments, the population of T cells in the first co-culturingstep is present in a population of PBMCs.

Exemplary methods for preparing T cells for TCR cloning or for preparingtumor antigen-specific T cells are illustrated in FIGS. 4, 7 and 15A-15Band described in Examples 2-3.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: (a) contacting apopulation of DCs derived from a population of PBMCs from an individualwith a target tumor antigen peptide to obtain antigen-loaded DCs; (b) afirst co-culture step comprising co-culturing a population of T cells(e.g., present in PBMCs) and a first population of the antigen-loadedDCs in an initial first co-culture medium comprising one or morecytokines (such as IL-2 or a plurality of cytokines) and an immunecheckpoint inhibitor (e.g., anti-PD-1 antibody); (c) an enrichment stepcomprising contacting the first co-culture with PBMCs loaded with thetarget tumor antigen peptide to obtain a stimulated co-culture, andisolating an enriched population of activated T cells from thestimulated co-culture using a ligand that specifically recognizes acytokine (such as IFNy) to obtain an enriched population of activated Tcells; (d) a second co-culture step comprising co-culturing the enrichedpopulation of activated T cells and a second population of theantigen-loaded DCs in an initial second co-culture medium comprising oneor more cytokines (such as IL-2 or a plurality of cytokines) and animmune checkpoint inhibitor (e.g., anti-PD-1 antibody) to obtain asecond co-culture, and adding an anti-CD3 antibody (e.g., OKT3) to thesecond co-culture about 1 day to about 3 days (e.g., about 2 days) afterthe second co-culture starts, thereby providing a population of T cellsfor TCR cloning, wherein the population of T cells in the firstco-culturing step is obtained from an individual who has clinicallybenefitted from a MASCT, and wherein at least about 10% (e.g., at least20% or 50%) of the population of T cells for TCR cloning specificallyresponds to the target tumor antigen peptide. In some embodiments, theantigen-loaded DCs are cultured in a DC maturation medium comprising atoll-like receptor (TLR) agonist. In some embodiments, the DC maturationmedium comprises INFy, MPLA and PGE2. In some embodiments, theantigen-loaded DCs are cultured in the DC maturation medium for about 8to about 12 days. In some embodiments, the ratio between the populationof T cells and the first population of the antigen-loaded DCs is about20:1. In some embodiments, the population of T cells and the populationof antigen-loaded DCs are co-cultured for about 2-3 days. In someembodiments, the ratio between the enriched population of activated Tcells and the second population of antigen-loaded DCs is about 1:1. Insome embodiments, the enriched population of activated T cells and thesecond population of antigen-loaded DCs are co-cultured for about 15-20days (e.g., about 16 days). Exemplary methods are shown in FIGS. 4 and 7.

In some embodiments, there is provided a method of preparing apopulation of T cells for TCR cloning, comprising: (a) co-culturing apopulation of tumor-antigen specific T cells with a first population ofAPCs (e.g., PBMCs, DCs, or cell line APCs) loaded with a target tumorantigen peptide for about 5 to 9 days (e.g., about 7 days) in aco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines) and an anti-CD3 antibody to obtain a firstpopulation of tumor antigen-specific T cells; and (b) co-culturing thefirst population of tumor antigen-specific T cells with a secondpopulation of APCs loaded with the target tumor antigen peptide forabout 5 to 9 days (e.g., about 7 days), thereby providing a secondpopulation of tumor antigen-specific T cells for TCR cloning, whereinthe population of tumor antigen-specific T cells is obtained from anindividual who has clinically benefitted from a MASCT, and wherein atleast about 10% (e.g., at least 20% or 50%) of the population of T cellsfor TCR cloning specifically responds to the target tumor antigenpeptide. In some embodiments, the stimulation step is repeated once ortwice. In some embodiments, the method further comprises: co-culturingthe second population of tumor antigen-specific T cells with a thirdpopulation of APCs loaded with the tumor antigen-specific T cells forabout 5 to 9 days (e.g., about 7 days), thereby providing a thirdpopulation of tumor antigen-specific T cells. In some embodiments, theAPCs are LCL cells with or without feeder cells. In some embodiments,the APCs are DCs. In some embodiments, the co-culture medium comprisesIL-2, IL-7, IL-15 and OKT3. In some embodiments, the ratio between theantigen-loaded APCs and the first, second or third population of tumorantigen-specific T cells is about 1:1 to about 1:10 (e.g., about 1:4).Exemplary methods are shown in FIGS. 15A-15B.

The tumor antigen-specific T cells used for the sequencing step have ahigh percentage of T cells that specifically responds to the targettumor antigen peptide or epitope thereof. For example, at least aboutany one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher of the tumorantigen-specific T cells used for the sequencing step specificallyrespond to the target tumor antigen peptide or epitope thereof. In someembodiments, any one of about 20-90%, 20-50%, 50-95%, 20-80%, 50-70%,40-60%, or 40%-80% of the tumor antigen-specific T cells used for thesequencing step specifically respond to the target tumor antigen peptideor epitope thereof. Because T cells express a large number of TCRα andTCRβ clonotypes, a high percentage of T cells that can specificallyrespond to the tumor antigen peptide is important for obtainingsuccessful cognate pairing information of the TCRα and TCRβ genes.

In some embodiments, the tumor antigen-specific T cells in anyembodiment of the isolated population of cells are capable of elicitingspecific immune response to the one or more tumor antigen peptides invivo or ex vivo. In some embodiments, the tumor antigen-specific T cellsare capable of increasing cytotoxic T cell activity in a humanindividual against more than one tumor antigen peptides. In someembodiments, the tumor antigen-specific T cells are characterized byhigh expression or secretion level of pro-inflammatory signal molecules,upon stimulation by the one or more tumor antigen peptides. In someembodiments, the expression or secretion level is determined bycomparing the expression or secretion level of a molecule (such as apro-inflammatory signal molecule) of the tumor antigen-specific T cellsupon stimulation with the one or more tumor antigen peptides to theexpression or secretion level upon stimulation with an irrelevantpeptide. In some embodiments, the control expression or secretion levelof a molecule is the expression or secretion level of the molecule in acontrol population of T cells measured under the same assay conditions.In some embodiments, the control population of T cells is a populationof T cells induced by one or more irrelevant peptides (such as peptidesnot corresponding to T cell receptor antigens, or random peptides). Insome embodiments, the control expression or secretion level of amolecule is an average or median expression or secretion level of themolecule in a plurality of control populations of T cells. In someembodiments, a high level of expression or secretion of a molecule inthe tumor antigen-specific T cells is at least about any of 1.5, 2, 2.5,3, 4, 5, 10, 20, 50, 100, 1000, or more times of the control expressionor secretion level.

In some embodiments, upon stimulation with the target tumor antigenpeptide, the tumor antigen-specific T cells express a plurality ofpro-inflammatory molecules, such as IFNγ, TNFα, granzyme B, perforin, orany combination thereof. In some embodiments, at least about any one of10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher percentage oftumor antigen-specific T cells that secrete INF-y upon stimulation withthe target tumor antigen peptide. In some embodiments, at least aboutany one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higherpercentage of tumor antigen-specific T cells secrete TNF-α uponstimulation with the target tumor antigen peptide.

In some embodiments, the tumor antigen-specific T cells are prepared bysteps comprising: (a) contacting a population of DCs derived from apopulation of PBMCs from an individual with a target tumor antigenpeptide to obtain a first population of antigen-loaded DCs; (b) a firstco-culture step comprising co-culturing a population of T cells (e.g.,present in PBMCs) and the first population of antigen-loaded DCs in aninitial first co-culture medium comprising one or more cytokines (suchas IL-2 or a plurality of cytokines) and an immune checkpoint inhibitor(e.g., anti-PD-1 antibody), and adding an anti-CD3 antibody to the firstco-culture no more than about 7 days (e.g., about 5 days) after thefirst-co-culture starts to obtain a first co-culture; (c) an enrichmentstep comprising contacting the first co-culture with PBMCs loaded withthe target tumor antigen peptide to obtain a stimulated co-culture, andisolating an enriched population of activated T cells from thestimulated co-culture using a ligand that specifically recognizes acytokine (such as IFNy) to obtain an enriched population of activated Tcells; (d) a second co-culture step comprising co-culturing the enrichedpopulation of activated T cells and a second population of DCs loadedwith the target tumor antigen peptide in a co-culture medium comprisingone or more cytokines (such as IL-2 or a plurality of cytokines), animmune checkpoint inhibitor (e.g., anti-PD-1 antibody) and an anti-CD3antibody, thereby providing the tumor antigen-specific T cells. In someembodiments, the ratio between the population of T cells and the firstpopulation of antigen-loaded DCs is about 20:1. In some embodiments, thepopulation of T cells and the population of antigen-loaded DCs areco-cultured for about 13-14 days. In some embodiments, the ratio betweenthe enriched population of activated T cells and the second populationof antigen-loaded DCs is about 2:1. In some embodiments, the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs are co-cultured for about 9-13 days.

In some embodiments, the tumor antigen-specific T cells are prepared bysteps comprising: (a) contacting a population of DCs derived from apopulation of PBMCs from an individual with a target tumor antigenpeptide to obtain antigen-loaded DCs; (b) a first co-culture stepcomprising co-culturing a population of T cells (e.g., present in PBMCs)and a first population of the antigen-loaded DCs in an initial firstco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody); (c) an enrichment step comprising contacting thefirst co-culture with PBMCs loaded with the target tumor antigen peptideto obtain a stimulated co-culture, and isolating an enriched populationof activated T cells from the stimulated co-culture using a ligand thatspecifically recognizes a cytokine (such as IFNy) to obtain an enrichedpopulation of activated T cells; (d) a second co-culture step comprisingco-culturing the enriched population of activated T cells and a secondpopulation of the antigen-loaded DCs in a co-culture medium comprisingone or more cytokines (such as IL-2 or a plurality of cytokines), animmune checkpoint inhibitor (e.g., anti-PD-1 antibody) and an anti-CD3antibody, thereby providing the tumor antigen-specific T cells. In someembodiments, the antigen-loaded DCs are cultured in a DC maturationmedium comprising a toll-like receptor (TLR) agonist. In someembodiments, the DC maturation medium comprises INFy, MPLA and PGE2. Insome embodiments, the antigen-loaded DCs are cultured in the DCmaturation medium for about 8 to about 12 days. In some embodiments, theratio between the population of T cells and the first population of theantigen-loaded DCs is about 15:1. In some embodiments, the population ofT cells and the population of antigen-loaded DCs are co-cultured forabout 3-4 days. In some embodiments, the ratio between the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs is about 2:1. In some embodiments, the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs are co-cultured for about 13-23 days.

In some embodiments, the tumor antigen-specific T cells are prepared bysteps comprising: (a) contacting a population of DCs derived from apopulation of PBMCs from an individual with a target tumor antigenpeptide to obtain antigen-loaded DCs; (b) a first co-culture stepcomprising co-culturing a population of T cells (e.g., present in PBMCs)and a first population of the antigen-loaded dendritic cells in aninitial first co-culture medium comprising one or more cytokines (suchas IL-2 or a plurality of cytokines) and an immune checkpoint inhibitor(e.g., anti-PD-1 antibody); (c) an enrichment step comprising contactingthe first co-culture with PBMCs loaded with the target tumor antigenpeptide to obtain a stimulated co-culture, and isolating an enrichedpopulation of activated T cells from the stimulated co-culture using aligand that specifically recognizes a cytokine (such as IFNy) to obtainan enriched population of activated T cells; (d) a second co-culturestep comprising co-culturing the enriched population of activated Tcells and a second population of the antigen-loaded DCs in an initialsecond co-culture medium comprising one or more cytokines (such as IL-2or a plurality of cytokines) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody) to obtain a second co-culture, and adding ananti-CD3 antibody (e.g., OKT3) to the second co-culture about 1 day toabout 3 days (e.g., about 2 days) after the second co-culture starts,thereby providing the tumor antigen-specific T cells. In someembodiments, the antigen-loaded DCs are cultured in a DC maturationmedium comprising a toll-like receptor (TLR) agonist. In someembodiments, the DC maturation medium comprises INFy, MPLA and PGE2. Insome embodiments, the antigen-loaded DCs are cultured in the DCmaturation medium for about 8 to about 12 days. In some embodiments, theratio between the population of T cells and the first population of theantigen-loaded DCs is about 20:1. In some embodiments, the population ofT cells and the population of antigen-loaded DCs are co-cultured forabout 2-3 days. In some embodiments, the ratio between the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs is about 1:1. In some embodiments, the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs are co-cultured for about 15-20 days (e.g., about 16days).

In some embodiments, the tumor antigen-specific T cells are prepared bysteps comprising: (a) contacting a population of DCs derived from apopulation of PBMCs from an individual with a target tumor antigenpeptide to obtain antigen-loaded DCs; (b) a first co-culture stepcomprising co-culturing a population of T cells (e.g., present in PBMCs)and a first population of the antigen-loaded DCs in an initial firstco-culture medium comprising one or more cytokines (such as IL-2 or aplurality of cytokines) and an immune checkpoint inhibitor (e.g.,anti-PD-1 antibody); (c) an enrichment step comprising contacting thefirst co-culture with PBMCs loaded with the target tumor antigen peptideto obtain a stimulated co-culture, and isolating an enriched populationof activated T cells from the stimulated co-culture using a ligand thatspecifically recognizes a cytokine (such as IFNy) to obtain an enrichedpopulation of activated T cells; (d) a second co-culture step comprisingco-culturing the enriched population of activated T cells and a secondpopulation of the antigen-loaded DCs in an initial second co-culturemedium comprising one or more cytokines (such as IL-2 or a plurality ofcytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody)to obtain a second co-culture, and adding an anti-CD3 antibody (e.g.,OKT3) to the second co-culture about 1 day to about 3 days (e.g., about2 days) after the second co-culture starts, thereby providing the tumorantigen-specific T cells. In some embodiments, the antigen-loaded DCsare cultured in a DC maturation medium comprising a toll-like receptor(TLR) agonist. In some embodiments, the DC maturation medium comprisesINFy, MPLA and PGE2. In some embodiments, the antigen-loaded DCs arecultured in the DC maturation medium for about 8 to about 12 days. Insome embodiments, the ratio between the population of T cells and thefirst population of the antigen-loaded DCs is about 20:1. In someembodiments, the population of T cells and the population ofantigen-loaded DCs are co-cultured for about 2-3 days. In someembodiments, the ratio between the enriched population of activated Tcells and the second population of antigen-loaded DCs is about 1:1. Insome embodiments, the enriched population of activated T cells and thesecond population of antigen-loaded DCs are co-cultured for about 15-20days (e.g., about 16 days).

In some embodiments, the method uses PBMC obtained from an individualwho has previously received an immunotherapy (e.g., MASCT) to preparetumor-antigen specific T cells used in the sequencing step.

In some embodiments, the method comprises: a) contacting a firstpopulation of PBMCs from the individual with the target tumor antigenpeptide to provide a population of PBMCs loaded with the target tumorantigen peptide; b) subjecting the population of PBMCs loaded with thetarget tumor antigen peptide to an enrichment process to provide anenriched population of activated T cells; c) optionally contacting apopulation of APCs (e.g., PBMCs, or DCs) with the target tumor antigenpeptide to provide a population of antigen-loaded APCs; d) aco-culturing step, comprising co-culturing the enriched population ofactivated T cells with the population of antigen-loaded APCs to obtain apopulation of tumor antigen-specific T cells. In some embodiments, thePBMCs are contacted with the target tumor antigen peptide for no morethan about 5, 4, 3, 2, or 1 day prior to the enrichment process. In someembodiments, the enrichment process comprises contacting the firstco-culture with PBMCs loaded with the target tumor antigen peptide toobtain a stimulated co-culture, and isolating an enriched population ofactivated T cells from the stimulated co-culture using a ligand thatspecifically recognizes a cytokine (such as IFNy) or a cell surfacemolecule. In some embodiments, the co-culturing step comprisesco-culturing the enriched population of activated T cells with thepopulation of antigen-loaded APCs in a co-culture medium comprising oneor more cytokines (such as IL-2 or a plurality of cytokines), an immunecheckpoint inhibitor, and an anti-CD3 antibody. In some embodiments, theco-culturing step comprises co-culturing the enriched population ofactivated T cells with the population of antigen-loaded APCs in aninitial co-culture medium comprising one or more cytokines (such as IL-2or a plurality of cytokines) and an immune checkpoint inhibitor toprovide a co-culture, and adding an anti-CD3 antibody to the co-culture.In some embodiments, the anti-CD3 antibody is added to the co-culture atabout 1-3 days after the co-culturing starts. In some embodiments, theenriched population of activated T cells and the population ofantigen-loaded APCs are co-cultured for a total of about 12-25 days.

In some embodiments, the PBMCs are freshly obtained. In someembodiments, the PBMCs are obtained by thawing a frozen stock of PBMCs.In some embodiments, the PBMCs are autologous, i.e. obtained from theindividual being treated. In some embodiments, the PBMCs are contactedwith cytokines, such as IL-2, GM-CSF, or the like, to inducedifferentiation, maturation, or proliferation of certain cells (such asDCs, T cells, or combination thereof) in the PBMCs concurrently or afterthe contacting step.

In some embodiments, the tumor antigen-specific T cells are prepared bysteps comprising: (a) contacting a population of PBMCs with a targettumor antigen peptide to obtain a population of stimulated PBMCs; (b)isolating an enriched population of activated T cells from thepopulation of stimulated PBMCs using a ligand that specificallyrecognizes a cytokine (such as IFNy) to obtain an enriched population ofactivated T cells; and (c) a co-culture step comprising co-culturing theenriched population of activated T cells and a population of DCs loadedwith a target tumor antigen peptide in an initial co-culture mediumcomprising one or more cytokines (such as IL-2 or a plurality ofcytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody)to obtain a first co-culture, and adding an anti-CD3 antibody (e.g.,OKT3) to the first co-culture about 1 day to about 3 days (e.g., about 1day or 2 days) after the first co-culture starts, thereby providing thetumor antigen-specific T cells. In some embodiments, the PBMCs are froma frozen stock. In some embodiments, the PBMCs are freshly obtained fromthe individual. In some embodiments, the antigen-loaded DCs are preparedby contacting a population of PBMCs with a target tumor antigen peptide.In some embodiments, the ratio between the enriched population ofactivated T cells and the population of antigen-loaded DCs is about 1:1.In some embodiments, the enriched population of activated T cells andthe population of antigen-loaded DCs are co-cultured for about 7 toabout 21 days.

Co-Culturing

The methods described herein and the MASCT methods comprise one or more(such as 1, 2, 3, or more) co-culturing steps. In some embodiments, themethod comprises a first co-culturing step, comprising co-culturing apopulation of T cells with a population of DCs loaded with the targettumor antigen peptide. In some embodiments, in the first co-culturingstep, the population of T cells is co-cultured with the first populationof antigen-loaded DCs for no more than about 7 days, such as about anyone of 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, the populationof T cells is co-cultured with the first population of antigen-loadedDCs for about 1-3 days, such as about 2-3 days.

In some embodiments, the first co-culturing step comprises co-culturinga first population of antigen-loaded DCs and the population of T cellsin a first co-culture medium comprising one or more cytokines (such as aplurality of cytokines) and an immune checkpoint inhibitor. In someembodiments, the first co-culture medium comprises an anti-CD3 antibody.In some embodiments, the first co-culture medium does not comprise ananti-CD3 antibody. In some embodiments, the first co-culturing stepcomprises co-culturing a first population of antigen-loaded DCs and thepopulation of T cells in a first initial co-culture medium comprisingone or more cytokines (such as a plurality of cytokines) and an immunecheckpoint inhibitor to provide a first co-culture; and adding ananti-CD3 antibody to the first co-culture.

In some embodiments, the method comprises a second co-culturing step,comprising co-culturing an enriched population of activated T cells witha population of DCs loaded with the target tumor antigen peptide. Insome embodiments, in the second co-culturing step, the enrichedpopulation of activated T cells and the second population ofantigen-loaded DCs are co-cultured for a total of at least about any oneof 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 days. Insome embodiments, the enriched population of activated T cells isco-cultured with the second population of antigen-loaded DCs for about12 days to about 25 days, such as about any one of 12-15, 15-18, 18-21,15-20, 20-25, 15, 18, 19, 20, 21, or 22 days.

In some embodiments, the enriched population of activated T cells andthe second population of antigen-loaded DCs are co-cultured in thepresence of the anti-CD3 antibody for at least about any one of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25 or more days. In some embodiments,the enriched population of activated T cells and the second populationof antigen-loaded DCs are co-cultured in the presence of the anti-CD3antibody for about any one of 8-18, 10-20, 1-25, or 12-25 days. In someembodiments, the enriched population of activated T cells and the secondpopulation of antigen-loaded DCs are initially co-cultured without ananti-CD3 antibody for about 1-5 days, such as about 1, 2, or 3 days.

In some embodiments, the second co-culturing step comprises co-culturinga second population of antigen-loaded DCs and the enriched population ofactivated T cells in a second co-culture medium comprising one or morecytokines (e.g., a plurality of cytokines), an immune checkpointinhibitor. In some embodiments, the second co-culture medium comprisesan anti-CD3 antibody. In some embodiments, the second co-culture mediumdoes not comprise an anti-CD3 antibody. In some embodiments, the secondco-culturing step comprises co-culturing a second population ofantigen-loaded DCs and the enriched population of activated T cells in asecond initial co-culture medium comprising one or more cytokines (e.g.,a plurality of cytokines) and an immune checkpoint inhibitor to providea second co-culture; and adding an anti-CD3 antibody to the secondco-culture.

In some embodiments, the method of preparing tumor antigen-specific Tcells comprises: (1) a first co-culturing step, comprising co-culturinga population of T cells with a first population of DCs loaded with aplurality of tumor antigen peptides, and (2) a second co-culturing step,comprising co-culturing an enriched population of activated T cells witha second population of DCs loaded with one or more tumor antigenpeptides from the plurality of tumor antigen peptides.

In some embodiments, the method comprises a third co-culturing step,comprising co-culturing a population of tumor antigen-specific T cellswith a population of APCs (e.g., PBMCs such as fixed PBMCs, DCs, or cellline APCs such as LCL) loaded with the target tumor antigen peptide (oran epitope thereof). In some embodiments, the third co-culturing step isrepeated for one or more times (such as 1, 2, 3, 4, 5, 6 or more) timesto obtain further populations of tumor antigen-specific T cells. In someembodiments, repeating the third co-culturing step comprisingco-culturing a portion of the tumor antigen-specific T cells obtainedfrom the third co-culturing step with a second population ofantigen-loaded APCs (e.g., PBMCs such as fixed PBMCs, DCs, or cell lineAPCs such as LCL). In some embodiments, repeating the third co-culturingstep comprising adding to the third co-culture a fresh population ofantigen-loaded APCs (e.g., PBMCs such as fixed PBMCs, DCs, or cell lineAPCs such as LCL) at an interval of every about 5-9 days (e.g., about 7days).

In some embodiments, in the third co-culturing step, the population oftumor antigen-specific T cells and the population of antigen-loaded APCs(e.g., PBMCs such as fixed PBMCs, DCs, or cell line APCs such as LCL)are co-cultured for at least about any one of 2, 4, 6, 8, 10, 12, 14,16, 18, 20, or 14 days. In some embodiments, the population of tumorantigen-specific T cells and the population of antigen-loaded APCs(e.g., PBMCs such as fixed PBMCs, DCs, or cell line APCs such as LCL)are co-cultured for about 5 days to about 15 days, such as about any oneof 5-9, 7-10, 10-12, 12-15, 7, 8, 9, 10, 11, 12, 13, or 15 days.

In some embodiments, the third co-culturing step comprises co-culturinga population of antigen-loaded APCs (e.g., PBMCs such as fixed PBMCs,DCs, or cell line APCs such as LCL) and a population of tumorantigen-specific T cells in a third co-culture medium comprising one ormore cytokines (e.g., a plurality of cytokines) and an immune checkpointinhibitor. In some embodiments, the third co-culture medium does notcomprise an anti-CD3 antibody. In some embodiments, the third co-culturemedium comprises an anti-CD3 antibody. In some embodiments, the thirdco-culturing step comprises co-culturing a population of antigen-loadedAPCs (e.g., PBMCs such as fixed PBMCs, DCs, or cell line APCs such asLCL) and a population of tumor antigen-specific T cells in a thirdinitial co-culture medium comprising one or more cytokines (e.g., aplurality of cytokines) and an immune checkpoint inhibitor to provide athird co-culture; and adding an anti-CD3 antibody to the thirdco-culture.

The co-culture medium or the initial co-culture medium for eachco-culturing step may be the same or different. Unless indicatedotherwise, “co-culture medium” as discussed in the subsection“Co-culturing” includes the first, second and third co-culture medium;“Initial co-culture medium” as discussed in this subsection includes thefirst, second and third initial co-culture medium. In some embodiments,the co-culture medium (including the initial co-culture medium)comprises one or more (e.g., 1, 2, 3, 4, 5, or more) cytokines. In someembodiments, the co-culture medium (including the initial co-culturemedium) comprises a plurality of cytokines (also referred herein as“cytokine cocktail”). Exemplary cytokines include, but are not limitedto, IL-2, IL-7, IL-15, IL-21 and the like. In some embodiments, theco-culture medium (including the initial co-culture medium) comprisesIL-2. In some embodiments, the co-culture medium (including the initialco-culture medium) comprises IL-2, IL-7, IL-15 and IL-21. In someembodiments, the IL-2 is present at a concentration of at least aboutany of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,5000, 6000 or higher IU/ml in the co-culture medium (including theinitial co-culture medium). In some embodiments, the IL-2 is present ata concentration of no more than about any one of 1000, 500, 200, 100,50, 20, or lower IU/ml in the co-culture medium (including the initialco-culture medium). In some embodiments, the first co-culture mediumcomprises IL-2 at a concentration of no more than about 200 IU/mL (suchas about 150, 100 or 50 IU/ml). In some embodiments, the secondco-culture medium comprises IL-2 at a concentration of at least about2000 IU/mL (such as about 3000, 5000, or 6000 IU/mL). In someembodiments, the IL-7 is present at a concentration of at least aboutany one of 1, 2, 5, 10, 20, 50 or 100 ng/mL in the co-culture medium(including the initial co-culture medium). In some embodiments, theIL-15 is present at a concentration of at least about any one of 1, 2,5, 10, 20, 50 or 100 ng/mL in the co-culture medium (including theinitial co-culture medium). The cytokines may facilitate activation,maturation, and/or proliferation of the T cells, to prime T cells forlater differentiation into memory T cells, and/or suppress thepercentage of T_(REG) in the co-culture.

In some embodiments, the co-culture medium (including the initialco-culture medium) comprises one or more (such as any of 1, 2, 3, ormore) immune checkpoint inhibitors. Any known immune checkpointinhibitors may be used. In some embodiments, the immune checkpointinhibitor is a natural or engineered ligand of an inhibitory immunecheckpoint molecule, including, for example, ligands of CTLA-4 (e.g.,B7.1, B7.2), ligands of TIM-3 (e.g., Galectin-9), ligands of A2aReceptor (e.g., adenosine, Regadenoson), ligands of LAG-3 (e.g., MHCclass I or MHC class II molecules), ligands of BTLA (e.g., HVEM, B7-H4),ligands of KIR (e.g., MHC class I or MHC class II molecules), ligands ofPD-1 (e.g., PD-L1, PD-L2), ligands of IDO (e.g., NKTR-218, Indoximod,NLG919), and ligands of CD47 (e.g., SIRP-alpha receptor). The immunecheckpoint inhibitors may be of any suitable molecular modality,including, but not limited to, small molecules, nucleic acids (such asDNA, RNAi, or aptamer), peptides, or proteins (such as antibodies).

In some embodiments, the immune checkpoint inhibitor is an antibody(such as antagonist antibody) that targets an inhibitory immunecheckpoint protein selected from the group consisting of anti-CTLA-4(e.g., Ipilimumab, Tremelimumab, KAHR-102), anti-TIM-3 (e.g., F38-2E2,ENUM005), anti-LAG-3 (e.g., BMS-986016, IMP701, IMP321, C9B7W), anti-KIR(e.g., Lirilumab and IPH2101), anti-PD-1 (e.g., Nivolumab, Pidilizumab,Pembrolizumab, BMS-936559, atezolizumab, Pembrolizumab, MK-3475,AMP-224, AMP-514, STI-A1110, TSR-042, SHR1210), anti-PD-L1 (e.g.,KY-1003 (EP20120194977), MCLA-145, RG7446, BMS-936559, MEDI-4736,MSB0010718C, AUR-012, STI-A1010, PCT/US2001/020964, MPDL3280A, AMP-224,Dapirolizumab pegol (CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42(OSU-HDAC42,HDAC-42,AR42,AR 42,OSU-HDAC 42,OSU-HDAC-42,NSCD736012,HDAC-42,HDAC 42,HDAC42,NSCD736012,NSC-D736012), MEDI-9447),anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD47 (e.g., CC-90002,TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1,anti-B7-H4, anti-CD52 (such as alemtuzumab), anti-IL-10, anti-IL-35, andanti-TGF-β (such as Fresolumimab). In some embodiments, the antibody isa monoclonal antibody. In some embodiments, the antibody is afull-length antibody. In some embodiments, the antibody is anantigen-binding fragment selected from the group consisting of Fab,Fab′, F(ab′)₂, Fv, scFv, BiTE, nanobody, and other antigen-bindingsubsequences of the full length antibody or engineered combinationsthereof. In some embodiments, the antibody is a human antibody, ahumanized antibody, or a chimeric antibody. In some embodiments, theantibody is a bispecific or multispecific antibody.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-1. In some embodiments, the immune checkpoint inhibitor is ananti-PD-1 antibody. Exemplary anti-PD-1 antibodies include, but are notlimited to, Nivolumab, pembrolizumab, pidilizumab, BMS-936559, andatezolizumab, Pembrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110,TSR-042, and SHR-1210. In some embodiments, the immune checkpointinhibitor is nivolumab (for example, OPDIVO®). In some embodiments, theimmune checkpoint inhibitor is Pembrolizumab (for example, KEYTRUDA®).In some embodiments, the immune checkpoint inhibitor is SHR-1210. Insome embodiments, the initial co-culture medium comprises IL-2, IL-7,IL-15, IL-21 and an anti-PD-1 antibody (e.g., SHR-1210).

A suitable concentration of the immune checkpoint inhibitor (e.g.,anti-PD-1 antibody) in the co-culture medium (including the initialco-culture medium) include, but are not limited to, at least about anyof 1, 2, 5, 10, 15, 20, 25 or more µg/mL. In some embodiments, theimmune checkpoint inhibitor (e.g., anti-PD-1 antibody) is present in theco-culture medium (including the initial co-culture medium) is any oneof about 1 µg/mL to about 10 µg/mL, about 10 µg/mL to about 20 µg/mL,about 1 µg/mL to about 25 µg/mL, or about 5 µg/mL to about 20 µg/mL.

The anti-CD3 antibody may be present in the co-culture at the time theco-culturing starts, or added to the co-culture after the co-culturingof the antigen-loaded DCs and the T cells, the enriched activated Tcells, or the population of tumor antigen-specific T cells starts. Insome embodiments, the anti-CD3 antibody is included in the co-culturemedium (including the initial co-culture medium). In some embodiments,the initial co-culture medium does not comprise the anti-CD3 antibody.

In some embodiments, the anti-CD3 antibody is added to the secondco-culture comprising the enriched population of activated T cells andthe second population of antigen-loaded DCs at no more than about anyone of 5, 4, 3, 2, or 1 day(s) after the second co-culturing starts. Insome embodiments, the anti-CD3 antibody is added to the secondco-culture comprising the enriched population of activated T cells andthe second population of antigen-loaded DCs about 1, 2, or 3 days afterthe second co-culturing starts. Any suitable anti-CD3 antibody may beused, including, but not limited to OKT3.

The T cells (e.g., T cells, enriched population of activated T cells, ortumor antigen-specific T cells) and antigen-loaded APCs (such as PBMCs,DCs or cell line APCs) may be present in the co-cultures at anappropriate ratio in terms of the number of cells. In some embodiments,the ratio between the population of T cells to the first population ofantigen-loaded DCs in the first co-culturing step is no more than aboutany one of 30:1, 25:1, 20:1, 15:1, 10:1, 8:1, or 5:1. In someembodiments, the ratio between the population of T cells to the firstpopulation of antigen-loaded DCs in the first co-culturing step is atleast about any one of 5:1, 8:1, 10:1, 15:1, 20:1, 25:1, or more. Insome embodiments, the ratio between the population of T cells to thefirst population of antigen-loaded DCs in the first co-culturing step isany one of about 5:1 to about 10:1, about 5:1 to about 20:1, about 10:1to about 20:1, about 20:1 to about 30:1, or about 5:1 to about 30:1. Insome embodiments, the ratio between the enriched population of T cellsand the second population of antigen-loaded DCs is at least about anyone of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In someembodiments, the ratio between the enriched population of T cells andthe second population of antigen-loaded DCs is no more than about anyone of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1. In someembodiments, the ratio between the enriched population of T cells andthe second population of antigen-loaded DCs is any one of about 1:1 toabout 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 5:1to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about10:1 to about 20:1, about 1:1 to about 1:3, about 1:1 to about 2:1, orabout 2:1 to about 5:1. In some embodiments, the ratio between thepopulation of tumor antigen-specific T cells and the population ofantigen-loaded APCs (e.g., PBMCs such as fixed PBMCs, DCs, or cell lineAPCs) is at least about any one of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, or 10:1. In some embodiments, the ratio between the populationof tumor antigen-specific T cells and the population of antigen-loadedAPCs (e.g., PBMCs such as fixed PBMCs, DCs or cell line APCs) is no morethan about any one of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or1:1. In some embodiments, the ratio between the population of tumorantigen-specific T cells and the population of antigen-loaded APCs(e.g., PBMCs such as fixed PBMCs, DCs, or cell line APCs) is any one ofabout 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about5:1, about 5:1 to about 10:1, about 10:1 to about 15:1, about 15:1 toabout 20:1, about 10:1 to about 20:1, about 1:3 to about 3:1, about 1:1to about 3:1, about 1:1 to about 2:1, or about 2:1 to about 5:1.

In some embodiments, the T cells and the APCs (e.g., PBMCs, DCs, or cellline APCs) are derived from the same individual, e.g., the individualwho has clinically benefitted from the MASCT. In some embodiments, theAPCs (e.g., PBMCs, DCs, or cell line APCs) are not derived from theindividual who has clinically benefitted from the MASCT. In someembodiments, the T cells, the APCs (e.g., PBMCs, DCs, or cell line APCs)or both are derived from autologous sources, e.g., from the individualthat receives the engineered immune cells expressing the TCR. In someembodiments, the T cells, the APCs (e.g., PBMCs, DCs, or cell line APCs)or both are derived from allogenic sources.

In some embodiments, the T cells and/or the APCs (e.g., PBMCs or DCs)are obtained from an individual who has previously received animmunotherapy. In some embodiments, the individual is immunologicallyresponsive to the immunotherapy. “Immunologically responsive” to animmunotherapy means that the individual has developed specific immuneresponse to one or more tumor antigens in response to the immunotherapy.In some embodiments, the T cells and/or the APCs (e.g., PBMCs or DCs)are obtained from an individual who has clinically benefitted from theimmunotherapy. An individual who “clinically benefitted” from a therapyhas demonstrated a clinical response to the therapy as assessed by aphysician. Exemplary clinical responses include, but are not limited to,complete response (“CR”), partial response (“PR”), and stable disease(“SD”). Immunotherapies, include, but are not limited to, immunecheckpoint inhibitors, adoptive immune cell therapy (e.g., adoptive Tcell therapy, CIK, TIL, CAR-T, and TCR-T therapies), cancer vaccine,oncolytic viruses and combinations thereof. In some embodiments, the Tcells and/or the APCs (e.g., PBMCs or DCs) are obtained from anindividual who has previously received a MASCT. In some embodiments, theindividual is capable of developing a specific immune response against atumor antigen peptide in the MASCT. Specific immune response against atumor antigen peptide can be determined using known assays in the art,such as ELISPOT assays. In some embodiments, the individual hasclinically benefitted from the MASCT. In some embodiments, theindividual has tumor antigen-specific immune response(s). In someembodiments, the individual has tumor antigen-specific immune responsesand clinically benefitted from a MASCT.

The population of T cells used in any embodiment of the methodsdescribed herein may be derived from a variety of sources. A convenientsource of T cells is from the PBMCs of the human peripheral blood. Thepopulation of T cells may be isolated from the PBMCs, or alternatively,a population of PBMCs enriched with T cells (such as by addition of Tcell specific antibodies and cytokines) can be used in the co-culture.In some embodiments, the population of T cells used in the firstco-culturing step is obtained from the peripheral blood mononuclearcells (PBMCs). In some embodiments, the PBMCs are obtained by densitygradient centrifugation of a sample of peripheral blood. In someembodiments, the population of T cells used in the first co-culturingstep is present in the PBMCs.

Enrichment of Activated T Cells

The methods described herein comprise an enrichment step comprisingenriching activated T cells from a co-culture comprising a firstpopulation of antigen-loaded DCs and a population of T cells. In someembodiments, the method comprises an enrichment step comprisingenriching activated T cells from PBMCs stimulated with the target tumorantigen peptide or fragments thereof.

In some embodiments, the enrichment process comprises selectingactivated T cells based on one or more (such as any one of 1, 2, 3, ormore) biomarkers of T cell activation from the co-culture in response tostimulation by the target tumor antigen peptide or fragments thereof. Insome embodiments, APCs (such as PBMCs) loaded with the target tumorantigen peptide are used to stimulate the activated T cells in theco-culture. In some embodiments, the enrichment process comprisesisolating activated T cells expressing one or more biomarkers, such ascell surface molecules or secreted molecules, from the co-culture.

In some embodiments, the enrichment process comprises isolatingactivated T cells expressing or secreting one or more cytokines from theco-culture that has been stimulated by the target tumor antigen peptideor fragments thereof. In some embodiments, the enrichment step comprisescontacting the first co-culture with antigen-loaded PBMCs to obtain astimulated co-culture, and isolating an enriched population of activatedT cells from the stimulated co-culture using a ligand that specificallyrecognizes a cytokine. Exemplary cytokines include, but are not limitedto, IFNγ and TNFα. Ligands that specifically recognize the cytokine,such as antibodies or receptors for the cytokine, can be used to isolatethe enriched population of activated T cells. In some embodiments, theenrichment step comprises contacting the first co-culture withantigen-loaded PBMCs to obtain a stimulated co-culture, and isolating anenriched population of activated T cells from the stimulated co-cultureusing a ligand that specifically recognizes a cell surface molecule,such as 4-1BB (also known as CD137).

In some embodiments, the method comprises contacting the co-culture withPBMCs loaded with the target tumor antigen peptide or fragments thereofto obtain a stimulated co-culture, and isolating an enriched populationof activated T cells from the stimulated co-culture using a ligand thatspecifically recognizes a cytokine or a cell surface molecule. In someembodiments, the cytokine is IFNy. In some embodiments, the cell surfacemolecule is 4-1BB.

In some embodiments, the enrichment process comprises isolatingactivated T cells secreting IFNy from the co-culture upon stimulation bythe target tumor antigen peptide or fragments thereof. In someembodiments, the enrichment process comprises isolating CD3⁺IFNγ⁺ cellsfrom the co-culture upon stimulation by the target tumor antigen peptideor fragments thereof. In some embodiments, the enrichment processcomprises: (1) contacting the co-culture comprising a first populationof DCs loaded with the target tumor antigen peptide or fragments thereofand a population of T cells with the PBMCs loaded with the target tumorantigen peptide or fragments thereof for about 10-24 hours (such asabout 1 day) to obtain a stimulated co-culture; and (2) isolatingactivated T cells using a ligand that specifically recognizes IFNy fromthe stimulated co-culture. In some embodiments, the first population ofantigen-loaded DCs and the population of T cells have been co-culturedfor about 1-7 days (such as about 2-3 days) prior to the contacting withthe antigen-loaded PBMCs. In some embodiments, the co-culture and theantigen-loaded PBMCs are contacted for at least about any one of 2, 4,6, 12, 18, 24 or more hours prior to the isolating.

Activated T cells expressing a cytokine (such as IFNy) can be isolatedor enriched from the stimulated co-culture using any known methods inthe art. For example, commercial kits are available for isolating Tcells that secrete IFNy, including IFNy Secretion Assay-Cell Enrichmentand Detection Kit from Miltenyi Biotec. In some embodiments, theactivated T cells secreting IFNy are isolated by: (1) contacting theco-culture with an IFNy catch reagent that specifically binds to a cellsurface antigen on T cells and IFNy; (2) contacting the IFNy catchreagent treated co-culture with an anti-IFNy antibody (e.g., ananti-IFNy antibody conjugated to R-phycoerthrin or PE); (3) contactingthe anti-IFNy antibody treated co-culture with a magnetic beadcomprising a secondary antibody that recognizes the anti-IFNy antibody(e.g., an anti-PE antibody); and (4) isolating the magnetic beads usinga magnetic field (e.g., using a MACS™ separator column), therebyobtaining an enriched population of activated T cells.

In some embodiments, the activated T cells expressing a cell surfacebiomarker are isolated by: (1) contacting the co-culture with afluorescently labeled antibody against the cell surface biomarker; and(2) isolating cells bound to the fluorescently labeled antibody from theco-culture by flow cytometry.

Antigen Loading of APCs

The methods described herein and the MASCT methods use APCs (such asPBMCs, dendritic cells, or cell line APCs) loaded with one or more tumorantigen peptides. In some embodiments, the antigen-loaded APCs (e.g.,antigen-loaded DCs) are freshly prepared for one or more of theco-culturing steps. In some embodiments, the antigen-loaded APCs (e.g.,antigen-loaded DCs) are freshly prepared for each co-culturing step. Insome embodiments, the antigen-loaded APCs (e.g., antigen-loaded DCs) areprepared, cultured in a DC maturation medium, and used for one or moreco-culturing or stimulation steps. The antigen-loaded DCs used in thefirst, second and third co-culturing steps may be obtained from a singlebatch or separate batches of antigen-loaded DCs. Unless indicatedotherwise, the features described in this section for the APCs (e.g.,DCs) apply to all APCs (e.g., DCs) used in each of the co-culturingsteps; and the methods and features described in this section for theantigen-loaded APCs (e.g., DCs) apply to the first population, thesecond population, and the third population of antigen-loaded DCs andother types of APCs. APCs include, but are not limited to, PBMCs, DCs, Bcells, or macrophages. The APCs described herein can be primary cells orderived from cell lines. In some embodiments, the APCs are PBMCs. Insome embodiments, the APCs are fixed PBMCs. Fixing PBMCs can destroy theproliferation capacity of the PBMCs, while maintaining the antigenpresenting capacity of PBMCs.

The antigen-loaded DCs used in each co-culturing step may be loaded withthe same pool of tumor antigen peptides or different pool of tumorantigen peptides. In some embodiments, the first population of DCs inthe first co-culturing step is loaded with the same pool of tumorantigen peptides used to load the second population of DCs in the secondco-culturing step. In some embodiments, the second population of DCs inthe second co-culturing step is loaded with a subset of the pool oftumor antigen peptides used to load the first population of DCs in thefirst co-culturing step. In some embodiments, the third population ofDCs in the third co-culturing step is loaded with a subset of the poolof tumor antigen peptides used to load the first population of DCs inthe first co-culturing step and/or the second population of DCs in thesecond co-culturing step. In some embodiments, the subset of the pool oftumor antigen peptides includes fragments of the tumor antigen peptidesand combinations thereof. In some embodiments, a single tumor antigenpeptide (i.e., the target tumor antigen peptide) or fragment thereof isused to load the APCs (such as DCs) used in the second and thirdco-culturing steps.

In some embodiments, the first population of antigen-loaded DCs used inthe first co-culturing step is prepared using the plurality of tumorantigen peptides that the individual used in previous MASCTs. In someembodiments, the first population of antigen-loaded DCs used in thefirst co-culturing step is prepared using one or more tumor antigenpeptides that the individual has specific immune response to in previousMASCTs. In some embodiments, individual tumor antigen peptides from oneor more target tumor antigen peptides or fragments thereof, andcombinations thereof are screened (e.g., by ELISPOT) for specific immuneresponse by PBMCs, activated T cells, or tumor antigen-specific T cellsderived from an individual to identify one or more target tumor antigenpeptides (including fragments thereof) for use in subsequent preparationof tumor antigen-specific T cells.

In some embodiments, prior to each co-culturing step, the methodcomprises one or more of the following steps: (1) obtaining PBMCs froman individual; (2) obtaining a population of monocytes from the PBMCs;(3) inducing differentiation of the population of monocytes intoimmature DCs; (4) contacting the immature DCs with one or more tumorantigen peptides to obtain a population of antigen-loaded DCs; and (5)culturing the population of antigen-loaded DCs in a DC maturation mediumcomprising a TLR agonist (such as MPLA).

In some embodiments, the antigen-loaded DCs are prepared by: (a)contacting a population of DCs with one or more tumor antigen peptidesto obtain a population of antigen-loaded DCs, and (b) culturing thepopulation of antigen-loaded DCs in a DC maturation medium comprising atoll-like receptor (TLR) agonist. Exemplary TLR agonists include, butare not limited to, MPLA (monophosphoryl lipid A), Poly I:C, resquimod,gardiquimod, and CL075. Cytokines and other appropriate molecules, suchas INFγ and PGE2 (prostaglandin E2) may be further included in theculturing media in the maturation step.

In some embodiments, the antigen-loaded DCs are prepared by: (a)contacting a population of DCs with one or more tumor antigen peptidesto obtain a population of antigen-loaded DCs, and (b) culturing thepopulation of antigen-loaded DCs in a DC maturation medium comprisingMPLA, INFy and PGE2.

In some embodiments, the antigen-loaded DCs are prepared by: (a)inducing differentiation of a population of monocytes into immature DCs;(b) contacting a population of immature DCs with one or more tumorantigen peptides to obtain a population of antigen-loaded DCs; and (c)culturing the population of the antigen-loaded DCs in a DC maturationmedium comprising MPLA, INFy and PGE2. In some embodiments, thepopulation of monocytes is obtained from PBMCs.

In some embodiments, the antigen-loaded PBMCs are prepared by contactinga population of PBMCs with one or more tumor antigen peptides. In someembodiments, antigen-loaded cell line APCs are prepared by contacting apopulation of cell line APCs (e.g., LCL) with one or more tumor antigenpeptides.

The DC maturation medium may comprise a suitable concentration of MPLA,INFy and/or PGE2. In some embodiments, the DC maturation mediumcomprises MPLA at a concentration of at least about 0.5 µg/mL, such asat least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more µg/mL.In some embodiments, the DC maturation medium comprises MPLA at aconcentration of any one of about 0.5-10, 1-5, 5-10, or 2.5-7.5 µg/mL.In some embodiments, the DC maturation medium comprises INFy at aconcentration of at least about 100 IU/mL, such as at least about anyone of 150, 200, 250, 300, 400, 500, 600, 800, 1000 or more IU/mL. Insome embodiments, the DC maturation medium comprises INFy at aconcentration of about any one of 100-1000, 100-250, 250-500, 500-1000,or 250-750 IU/mL. In some embodiments, the DC maturation mediumcomprises PGE2 at a concentration of at least about 0.1 µg/mL, such asat least about any one of 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, or more µg/mL.In some embodiments, the DC maturation medium comprises PGE2 at aconcentration of about any one of 0.1-0.5, 0.1-0.3, 0.25-0.5 or 0.2-0.4µg/mL.

The immature DCs loaded with one or more tumor antigen peptides may beinduced by TLR agonists to mature for at least about any one of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, or 20 days. In some embodiments, the DCsloaded with one or more tumor antigen peptides are induced to mature forabout 8, 9, 10, 11, or 12 days.

In some embodiments, the antigen-loaded DCs are mature DCs that presentone or more tumor antigen peptides. The mature DCs prepared by any ofthe methods described herein may present at least about any one of 1, 5,10, 15, 20, 25, 30, 35, 40, 50 or more tumor antigen peptides. Comparedto naïve DCs, or DCs that have not been loaded with a plurality of tumorantigen peptides, the multiple-antigen loaded DCs may have enhancedlevel of presentation for at least about any of 1, 5, 10, 15, 20, 25,30, 35, 40, 50 or more tumor antigen peptides. In some embodiments, themature DCs have enhanced level of presentation for more than 10 tumorantigen peptides. In some embodiments, the mature DCs have enhancedlevel of presentation of about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, or more tumor antigen peptides derived from proteinsselected from the group consisting of hTERT, p53, Survivin, NY-ESO-1,CEA, CCND1, RGS5, MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3,CDCA1, WT1, KRAS, PARP4, MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6,HPV18-E7, HPV58-E6, HPV58-E7, HBcAg, HBV polymerase, GPC3, SSX, and AFP.

In some embodiments, the antigen-loaded APCs (e.g., DCs, PBMCs, or cellline APCs) are prepared by pulsing one or more tumor antigen peptidesinto a population of APCs. In some embodiments, the antigen-loaded DCsare prepared by pulsing one or more tumor antigen peptides into apopulation of DCs, such as immature DCs, or DCs contained in or derived(such as differentiated) from the PBMCs. As known in the art, pulsingrefers to a process of mixing cells, such as APCs (e.g., PBMCs or DCs,or cell line APCs), with a solution containing antigen peptides, andoptionally subsequently removing the antigen peptides from the mixture.The population of DCs may be contacted with one or more tumor antigenpeptides for seconds, minutes, or hours, such as about at least any oneof 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes,30 minutes, 1 hour, 5 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, one week, 10 days, or more. The concentration of each tumorantigen peptide used in the contacting step may be at least about anyone of 0.1, 0.5, 1, 2, 3, 5, or 10 µg/mL. In some embodiments, theconcentration of the tumor antigen peptides is about 0.1-200 µg/mL,including for example about any of 0.1-0.5, 0.5-1, 1-10, 10-50, 50-100,100-150, or 150-200 µg/mL.

In some embodiments, the population of APCs (e.g., DCs or PBMCs, or cellline APCs) is contacted with one or more tumor antigen peptides in thepresence of a composition that facilitates the uptake of the one or moretumor antigen peptides by the APCs (e.g., DCs or PBMCs, or cell lineAPCs). In some embodiments, compounds, materials or compositions may beincluded in a solution of the one or more tumor antigen peptides tofacilitate peptide uptake by the APCs (e.g., DCs or PBMCs, or cell lineAPCs). Compounds, materials or compositions that facilitate the uptakeof the one or more tumor antigen peptides by the APCs (e.g., DCs orPBMCs, or cell line APCs) include, but are not limited to, lipidmolecules and peptides with multiple positively charged amino acids. Insome embodiments, more than about any of 50%, 60%, 70%, 80%, 90%, or 95%of the tumor antigen peptides are uptaken by the population of APCs(e.g., DCs or PBMCs, or cell line APCs). In some embodiments, more thanabout any of 50%, 60%, 70%, 80%, 90%, or 95% of the APCs (e.g., DCs orPBMCs, or cell line APCs) in the population uptake at least one tumorantigen peptide.

Dendritic cells (such as immature DCs) may be obtained from varioussources, including autologous sources, i.e. from the individualreceiving the TCR treatment. A convenient source of DCs is the PBMCsfrom the peripheral blood. For example, monocytes, a type of white bloodcells, are abundant in PBMCs, comprising about 5-30% of total PBMCs.Monocytes can be induced to differentiate into DCs, such as immatureDCs, using cytokines. In some embodiments, the immature DCs are preparedby obtaining a population of PBMCs, obtaining a population of monocytesfrom the population of PBMCs, and contacting the population of monocyteswith one or more cytokines (e.g., a plurality of cytokines) to obtain apopulation of immature DCs. Exemplary cytokines that may be used toinduce differentiation of monocytes include, but are not limited to,GM-CSF and IL-4, with conditions (such as concentrations, temperature,CO₂ level etc.) known in the art.

The adherent fraction of PBMCs contains the majority of monocytes inPBMCs. In some embodiments, the monocytes from the adherent fraction ofPBMCs are contacted with cytokines to obtain a population of immatureDCs. PBMCs can be conveniently obtained by centrifugation of a sample ofperipheral blood, or using apheresis methods to collect from anindividual. In some embodiments, the population of PBMCs is obtained bydensity gradient centrifugation of a sample of human peripheral blood.In some embodiments, the sample is from the individual that receives themultiple-antigen loaded DCs, activated T cells, engineered immune cellsexpressing TCR, or other immunotherapeutic compositions prepared usingthe multiple-antigen loaded DCs.

Tumor Antigen Peptides

The methods described herein and the MASCT methods use one or more tumorantigen peptides (including the target tumor antigen peptide) to prepareantigen-loaded APCs (such as antigen-loaded DCs), activated T cells andtumor antigen-specific T cells that can trigger specific immune responseex vivo and in vivo. In some embodiments, the plurality of tumor antigenpeptides is a plurality of synthetic tumor antigen peptides. In someembodiments, the plurality of tumor antigen peptides is not obtainedfrom a cell sample, such as a lysed cell composition. As used herein,“one or more tumor antigen peptides from a plurality of tumor antigenpeptides” refers to a sub-selection or all tumor antigen peptides in theplurality of tumor antigen peptides, including fragments of the tumorantigen peptides and combinations thereof. The features and parametersdescribed in this subsection are applicable to the target tumor antigenpeptide(s).

In some embodiments, each tumor antigen peptide comprises at least aboutany one of 1, 2, 3, 4, 5, or 10 epitopes from a single protein antigen(including a neoantigen). In some embodiments, each tumor antigenpeptide in the plurality of tumor antigen peptides comprises at leastone epitope recognizable by a T cell receptor. In some embodiments, theplurality of tumor antigen peptides comprises at least one tumor antigenpeptide that comprises at least 2 epitopes from a single proteinantigen. The tumor antigen peptide can be a naturally derived peptidefragment from a protein antigen containing one or more epitopes, or anartificially designed peptide with one or more natural epitopesequences, wherein a linker peptide can optionally be placed in betweenadjacent epitope sequences. In some preferred embodiments, the epitopescontained in the same tumor antigen peptide are derived from the sameprotein antigen.

The tumor antigen peptide may contain at least one MHC-I epitope, atleast one MHC-II epitope, or both MHC-I epitope(s) and MHC-IIepitope(s). In some embodiments, the plurality of tumor antigen peptidescomprises at least one peptide comprising an MHC-I epitope. In someembodiments, the plurality of tumor antigen peptides comprises at leastone peptide comprising an MHC-II epitope. In some embodiments, at leastone tumor antigen peptide in the plurality of tumor antigen peptidescomprises both MHC-I and MHC-II epitopes.

Special design strategies can be applied to the sequence of the tumorantigen peptides (including neoantigen peptides) in order to optimizethe immune response to DCs loaded with the tumor antigen peptides.Typically, a peptide longer than the exact epitope peptide can increaseuptake of the peptide into DCs. In some embodiments, an MHC-I or MHC-IIepitope sequence is extended at the N terminus or the C terminus or bothtermini according to the natural sequence of the protein harboring theepitope to obtain an extended sequence, wherein the extended sequence isamenable for presentation by both class I and class II MHC molecules,and by different subtypes of MHC molecules in different individuals. Insome embodiments, the epitope sequence is extended at one or bothtermini by at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,15, or 20 amino acid residues to generate the extended epitope. In someembodiments, the peptides comprising an MHC-I or MHC-II epitope furthercomprise additional amino acids flanking the epitope at the N-terminus,the C-terminus, or both. In some embodiments, each tumor antigen peptidein the plurality of tumor antigen peptides is at least about any one of10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acidslong. Different tumor antigen peptides in the plurality of tumor antigenpeptides may have the same length, or different lengths. In someembodiments, the plurality of tumor antigen peptides is each about 20-40amino acids long.

In some embodiments, the amino acid sequences of one or more epitopepeptides used to design a tumor antigen peptide in the presentapplication are based on sequences known in the art or available inpublic databases, such as the Peptide Database (Vigneron N. et al.Cancer Immunity, 13:15 (2013)).

In some embodiments, the amino acid sequences of one or more epitopepeptides are predicted based on the sequence of the antigen proteinusing a bioinformatics tool for T cell epitope prediction. Exemplarybioinformatics tools for T cell epitope prediction are known in the art,for example, see Yang X. and Yu X. (2009) “An introduction to epitopeprediction methods and software” Rev. Med. Virol. 19(2): 77-96. In someembodiments, the sequence of the antigen protein is known in the art oravailable in public databases. In some embodiments, the sequence of theantigen protein is determined by sequencing a sample (such as a tumorsample) of the individual being treated.

The present application contemplates tumor antigen peptides derived fromany tumor antigens and epitopes known in the art, including neoantigensand neoepitopes, or specially developed or predicted usingbioinformatics tools by the inventors.

In some embodiments, the plurality of tumor antigen peptides comprises afirst core group of general tumor antigen peptides. In some embodiments,the plurality of tumor antigen peptides further comprises a second groupof cancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises one or more neoantigenpeptides. In some embodiments, neoantigen peptides are cancer-typespecific antigen peptides. In some embodiments, the plurality of tumorantigen peptides consists of the first core group of general tumorantigen peptides. In some embodiments, the plurality of tumor antigenpeptides consists of the first core group of general tumor antigenpeptides and the second group of cancer-type specific antigen peptides.In some embodiments, the plurality of tumor antigen peptides consists ofneoantigen peptides only. In some embodiments, the plurality of tumorantigen peptides comprises a first core group of general tumor antigenpeptides and one or more neoantigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides, a second group of cancer-type specificantigen peptides, and one or more neoantigen peptides.

In some embodiments, the plurality of tumor antigen peptides comprises afirst core group of general tumor antigen peptides. In some embodiments,the plurality of tumor antigen peptides further comprises a second groupof cancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises one or more neoantigenpeptides. In some embodiments, neoantigen peptides are cancer-typespecific antigen peptides. In some embodiments, the plurality of tumorantigen peptides consists of the first core group of general tumorantigen peptides. In some embodiments, the plurality of tumor antigenpeptides consists of the first core group of general tumor antigenpeptides and the second group of cancer-type specific antigen peptides.In some embodiments, the plurality of tumor antigen peptides consists ofneoantigen peptides only. In some embodiments, the plurality of tumorantigen peptides comprises a first core group of general tumor antigenpeptides and one or more neoantigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides, a second group of cancer-type specificantigen peptides, and one or more neoantigen peptides.

The first core group of general tumor antigen peptides is derived fromtumor antigens commonly overexpressed by a variety of cancers ofdifferent types. Therefore, the first core group of general tumorantigen peptides is useful to prepare dendritic cells and/or activated Tcells for treating individuals with different cancer types. For example,in some embodiments, the first core group of general tumor antigenpeptides is useful for methods described herein for treating a varietyof cancers, such as lung cancer, colon cancer, gastric cancer, prostatecancer, melanoma, lymphoma, pancreatic cancer, ovarian cancer, breastcancer, glioma, esophageal cancer, nasopharyngeal carcinoma, cervicalcancer, renal carcinoma, or hepatocellular carcinoma. Exemplary tumorantigen peptides of the first core group include, but are not limitedto, peptides derived from hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1,MET, MUC1, Her2, MAGEA1, MAGEA3, WT-1, RGS5, MMP7, VEGFR (such as VEGFR1and VEGFR2), and CDCA1. The first core group may comprise peptidesderived from at least about any one of 1, 2, 5, 10, 15, 20, 25, 30, 40,50, 60, 70, 80 or more tumor antigens. The first core group may compriseat least about any one of 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70,80 or more general tumor antigen peptides. In some embodiments, thefirst core group comprises more than one general tumor antigen peptides.In some embodiments, the first core group comprises about 10 to about 20general tumor antigen peptides.

The second group of cancer-type specific antigen peptides is derivedfrom tumor antigens that are overexpressed only in one or a limitednumber of cancer types. Therefore, the second group of cancer-typespecific antigen peptides is useful to prepare dendritic cells and/oractivated T cells for treating individuals with a particular type ofcancer. Exemplary cancer-type specific antigen peptides for treatinghepatocellular carcinoma (HCC) include, but are not limited to, peptidesderived from SSX, AFP, and GPC3. In some embodiments, one or more cancer-specific antigen peptide is a virus-specific antigen peptide derivedfrom a virus that can induce cancer, or relates to cancer development inthe individual when infecting the individual. In some embodiments, thevirus-specific antigen peptide is specific to the subtype of the virusinfecting the individual. Exemplary virus-specific antigen peptides fortreating an HCC patient with concurrent infection of HBV include, butare not limited to, peptides derived from HBV core antigen (HBcAg), andHBV DNA polymerase. In some embodiments, the second group comprisesvirus-specific antigen peptides derived from HBV antigens, wherein themethod is to treat hepatocellular carcinoma in an individual. In someembodiments, the second group comprises virus-specific antigen peptidesderived from HPV antigens, wherein the method is to treat cervicalcancer in an individual. In some embodiments, the second group comprisesvirus-specific antigen peptides derived from EBV antigens, wherein themethod is to treat nasopharyngeal carcinoma in an individual. The secondgroup of cancer-type specific antigen peptides may comprise peptidesderived from at least about any one of 1, 2, 5, 10, 15, 20, 25, 30, 40,50 or more cancer-type specific antigens. The second group ofcancer-type specific antigen peptides may comprise at least about anyone of 1, 2, 5, 10, 15, 20, 25, 30, 40, 50 or more cancer-type specificantigen peptides. In some embodiments, the second group comprises morethan one cancer-type specific antigen peptides. In some embodiments, thesecond group comprises about 1 to about 10 cancer-type specific antigenpeptides. In some embodiments, the type of cancer targeted by thecancer-type specific antigen peptides is selected from the groupconsisting essentially of hepatocellular carcinoma, cervical cancer,nasopharyngeal carcinoma, endometrial cancer, colorectal cancer, breastcancer, endometrial cancer, and lymphoma.

In some embodiments, the plurality of tumor antigen peptides comprisesone or more (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)neoantigen peptides. In some embodiments, the plurality of tumor antigenpeptides consists of neoantigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises neoantigen peptides and nogeneral tumor antigen peptides. In some embodiments, the plurality oftumor antigen peptides comprises one or more general tumor antigenpeptides and one or more neoantigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises one or more general tumorantigen peptides, one or more cancer-type specific antigen peptides, andone or more neoantigen peptides. The neoantigen peptides are derivedfrom neoantigens. Neoantigens are newly acquired and expressed antigenspresent in tumor cells of the individual, such as the individual beingtreated for cancer. In some embodiments, neoantigens are derived frommutant protein antigens that are only present in cancer cells, butabsent in normal cells. Neoantigens may be uniquely present in the tumorcells (such as all tumor cells or a portion of tumor cells) of theindividual being treated for cancer, or present in individuals havingsimilar types of cancer as the individual being treated. In someembodiments, the neoantigen is a clonal neoantigen. In some embodiments,the neoantigen is a subclonal neoantigen. In some embodiments, theneoantigen is present in at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or more tumor cells in the individual. In someembodiments, the neoantigen peptide comprises an MHC-I restrictedneoepitope. In some embodiments, the neoantigen peptide comprises anMHC-II restricted neoepitope. In some embodiments, the neoantigenpeptide is designed to facilitate presentation of the neoepitope by bothclass I and class II MHC molecules, for example, by extending theneoepitope at both the N- and the C- termini. Exemplary neoantigenpeptides include, but are not limited to, neoepitope derived from mutantKRAS (e.g., KRAS^(G12A)), PARP4 (e.g., PARP4^(T11701)), MLL3(e.g.,MLL3^(C988F)), and MTHFR (e.g., MTHFR^(A222V)).

Neoantigen peptides can be selected based on the genetic profile of oneor more tumor sites of the individual being treated, and neoantigens arenot expressed in normal tissues. In some embodiments, the geneticprofile of the tumor sample comprises sequence information of the fullgenome. In some embodiments, the genetic profile of the tumor samplecomprises sequence information of the exome. In some embodiments, thegenetic profile of the tumor sample comprises sequence information ofcancer-associated genes.

Neoantigen peptides suitable for use in the present application may bederived from any mutant proteins, such as those encoded by mutantcancer-associated genes, in the tumor cells. In some embodiments, theneoantigen peptide comprises a single neoepitope derived from acancer-associated gene. In some embodiments, the neoantigen peptidecomprises more than one (such as 2, 3, or more) neoepitope derived froma cancer-associated gene. In some embodiments, the neoantigen peptidecomprises more than one (such as 2, 3, or more) neoepitope derived frommore than one (such as 2, 3, or more) cancer-associated genes. In someembodiments, the plurality of tumor antigens comprises a plurality ofneoantigen peptides derived from a single cancer-associated gene. Insome embodiments, the plurality of tumor antigens comprises a pluralityof neoantigen peptides derived from more than one (such as any of 2, 3,4, 5, or more) cancer-associated genes.

Cancer-associated genes are genes that are overexpressed in cancercells, but expressed at low levels in normal cells. Exemplarycancer-associated genes include, but are not limited to, ABL1, AKT1,AKT2, AKT3, ALK, ALOX12B, APC, AR, ARAF, ARID1A, ARID1B, ARID2, ASXL1,ATM, ATRX, AURKA, AURKB, AXL, B2M, BAP1, BCL2, BCL2L1, BCL2L12, BCL6,BCOR, BCORL1, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BUB1B,CADM2, CARD11, CBL, CBLB, CCND1, CCND2, CCND3, CCNE1, CD274, CD58,CD79B, CDC73, CDH1, CDK1, CDK2, CDK4, CDK5, CDK6, CDK9, CDKN1A, CDKN1B,CDKN1C, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK2, CIITA, CREBBP, CRKL,CRLF2, CRTC1, CRTC2, CSF1R, CSF3R, CTNNB1, CUX1, CYLD, DDB2, DDR2,DEPDC5, DICER1, DIS3, DMD, DNMT3A, EED, EGFR, EP300, EPHA3, EPHA5,EPHA7, ERBB2, ERBB3, ERBB4, ERCC2, ERCC3, ERCC4, ERCC5, ESR1, ETV1,ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, FAM46C, FANCA, FANCC, FANCD2,FANCE, FANCF, FANCG, FAS, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FH, FKBP9,FLCN, FLT1, FLT3, FLT4, FUS, GATA3, GATA4, GATA6, GLI1, GLI2, GLI3,GNA11, GNAQ, GNAS, GNB2L1, GPC3, GSTM5, H3F3A, HNF1A, HRAS, ID3, IDH1,IDH2, IGF1R, IKZF1, IKZF3, INSIG1, JAK2, JAK3, KCNIP1, KDM5C, KDM6A,KDM6B, KDR, KEAP1, KIT, KRAS, LINC00894, LMO1, LMO2, LMO3, MAP2K1,MAP2K4, MAP3K1, MAPK1, MCL1, MDM2, MDM4, MECOM, MEF2B, MEN1, MET, MITF,MLH1, MLL (KMT2A), MLL2 (KTM2D), MPL, MSH2, MSH6, MTOR, MUTYH, MYB,MYBL1, MYC, MYCL1 (MYCL), MYCN, MYD88, NBN, NEGR1, NF1, NF2, NFE2L2,NFKBIA, NFKBIZ, NKX2-1, NOTCH1, NOTCH2, NPM1, NPRL2, NPRL3, NRAS, NTRK1,NTRK2, NTRK3, PALB2, PARK2, PAX5, PBRM1, PDCD1LG2, PDGFRA, PDGFRB, PHF6,PHOX2B, PIK3C2B, PIK3CA, PIK3R1, PIM1, PMS1, PMS2, PNRC1, PRAME, PRDM1,PRF1, PRKAR1A, PRKCI, PRKCZ, PRKDC, PRPF40B, PRPF8, PSMD13, PTCH1, PTEN,PTK2, PTPN11, PTPRD, QKI, RAD21, RAF1, RARA, RB1, RBL2, RECQL4, REL,RET, RFWD2, RHEB, RHPN2, ROS1, RPL26, RUNX1, SBDS, SDHA, SDHAF2, SDHB,SDHC, SDHD, SETBP1, SETD2, SF1, SF3B1, SH2B3, SLITRK6, SMAD2, SMAD4,SMARCA4, SMARCB1, SMC1A, SMC3, SMO, SOCS1, SOX2, SOX9, SQSTM1, SRC,SRSF2, STAG1, STAG2, STAT3, STAT6, STK11, SUFU, SUZ12, SYK, TCF3,TCF7L1, TCF7L2, TERC, TERT, TET2, TLR4, TNFAIP3, TP53, TSC1, TSC2,U2AF1, VHL, WRN, WT1, XPA, XPC, XPO1, ZNF217, ZNF708, and ZRSR2.

In some embodiments, the plurality of tumor antigen peptides comprisesat least one (such as at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more) tumor antigen peptide each comprisingone or more epitopes encoded by a cancer-associated gene selected fromthe group consisting of hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1,RGS5, MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3, CDCA1, WT1,KRAS, PARP4, MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7,HPV58-E6, HPV58-E7, HBcAg, HBV polymerase, GPC3, SSX, and AFP. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides. In some embodiments, the plurality of tumorantigen peptides comprises tumor antigen peptides derived from hTERT,p53, Survivin, NY-ESO-1, CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3, WT-1,RGS5, VEGFR1, VEGFR2, and CDCA1.

In some embodiments, the one or more tumor antigen peptides is presentin a composition having at least about any one of 95%, 96%, 97%, 98%,99%, 99.9% or higher percentage of the tumor antigen peptides. In someembodiments, the purity of the one or more tumor antigen peptides is atleast about 98%. In some embodiments, the solubility of the one or moretumor antigen peptides in the medium for pulsing the tumor antigenpeptides into the DCs is at least about any one of 80%, 85%, 90%, 95%,98%, 99%, 99.9% or higher. In some embodiments, the one or more tumorantigen peptides is about 100% soluble in the medium for pulsing thetumor antigen peptides into the APCs.

MASCT

The TCRs described herein are obtained from the PBMCs or T cells of anindividual who has clinically benefitted from a MASCT. In someembodiments, the individual has developed specific response to thetarget tumor antigen peptide(s) or fragments thereof used in the methodsdescribed herein, for example, as determined by ELISPOT.

As used herein, “MASCT” or “Multiple Antigen Specific Cell Therapy”refers to methods of adoptive T cells therapy comprising administeringto an individual an effective amount of activated T cells prepared byco-culturing a population of T cells with a population of DCs loadedwith a plurality of tumor antigen peptides. MASCT methods have beendescribed, for example, in International Patent Application PublicationNo. WO2016145578A1, International Patent Application Nos.PCT/CN2018/081338, and PCT/CN2019/080535, the contents of which areincorporated herein by reference in their entirety. First-generationMASCT, precision MASCT, PBMC-based MASCT, customized MASCT,neoantigen-based MASCT, improved MASCT, and combination therapy withMASCT (e.g., immune checkpoint inhibitor and MASCT) are all within thescope of MASCT of the present application. Any suitable features andparameters for preparation of antigen-loaded DCs, preparation ofactivated T cells, enrichment steps, and co-culturing steps described inthe present application or in International Patent ApplicationsWO2016145578A1, PCT/CN2018/081338 and PCT/CN2019/080535 may be combinedin a MASCT treatment.

The individual may have received a single type of MASCT, or acombination of different types of MASCT, for example, customized MASCTand improved MASCT. The individual may have received one or more cyclesof the MASCT. In some embodiments, the individual has received at leastabout any one of 2, 5, 10, 15, 20 or more cycles of MASCT. In someembodiments, the individual has received MASCT over at least about anyone of 3 months, 6 months, 9 months, 12 months, 2 years, 3 years orlonger.

In some embodiments, the MASCT comprises: (i) co-culturing a populationof DCs loaded with a plurality of tumor antigen peptides comprising thetarget tumor antigen peptide(s) and a population of T cells to obtain apopulation of activated T cells, and (ii) administering to theindividual an effective amount of the activated T cells. In someembodiments, the MASCT comprises administering to the individual aneffective amount of the DCs loaded with the plurality of tumor antigenpeptides.

In some embodiments, the MASCT comprises: (i) co-culturing a populationof DCs loaded with a plurality of tumor antigen peptides comprising thetarget tumor antigen peptide and a population of T cells in an initialco-culture medium comprising one or more cytokines (e.g., a plurality ofcytokines) and an immune checkpoint inhibitor to provide a co-culture;(ii) adding an anti-CD3 antibody to the co-culture at about 3 to 7 daysafter the co-culturing starts to obtain the population of activated Tcells; and (iii) administering to the individual an effective amount ofthe activated T cells. In some embodiments, the MASCT comprisesadministering to the individual an effective amount of the DCs loadedwith the plurality of tumor antigen peptides.

In some embodiments, the MASCT comprises: (i) contacting a population ofDCs with a plurality of tumor antigen peptides comprising the targettumor antigen peptide to obtain a population of DCs loaded with theplurality of tumor antigen peptides; (ii) culturing the population ofDCs loaded with the plurality of tumor antigen peptides in a DCmaturation medium comprising MPLA; (iii) co-culturing the population ofDCs loaded with the plurality of tumor antigen peptides and a populationof T cells to obtain a population of activated T cells, and (iv)administering to the individual an effective amount of the activated Tcells. In some embodiments, the DC maturation medium comprises INFγ,MPLA and PGE2. In some embodiments, the MASCT comprises administering tothe individual an effective amount of the DCs loaded with the pluralityof tumor antigen peptides.

In some embodiments, the MASCT comprises: (i) contacting a population ofDCs with a plurality of tumor antigen peptides comprising the targettumor antigen peptide to obtain a population of DCs loaded with theplurality of tumor antigen peptides; (ii) culturing the population ofDCs loaded with the plurality of tumor antigen peptides in a DCmaturation medium comprising MPLA; (iii) co-culturing the population ofDCs loaded with the plurality of tumor antigen peptides and a populationof T cells in an initial co-culture medium comprising one or morecytokines (e.g., a plurality of cytokines) and an immune checkpointinhibitor to provide a co-culture; (iv) adding an anti-CD3 antibody tothe co-culture at about 3 to 7 days after the co-culturing starts toobtain the population of activated T cells; and (V) administering to theindividual an effective amount of the activated T cells. In someembodiments, the DC maturation medium comprises INFγ, MPLA and PGE2. Insome embodiments, the MASCT comprises administering to the individual aneffective amount of the DCs loaded with the plurality of tumor antigenpeptides.

In some embodiments, the MASCT comprises administering to the individualan effective amount of activated T cells, wherein the activated T cellsare prepared by co-culturing a population of T cells with a populationof antigen presenting cells (such as DCs) loaded with a plurality oftumor antigen peptides. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theindividual has previously been administered an effective amount ofantigen presenting cells loaded with the plurality of tumor antigenpeptides. In some embodiments, the method comprises administering to theindividual an effective amount of antigen presenting cells (such as DCs)loaded with the plurality of tumor antigen peptides. In someembodiments, the antigen presenting cells are administered about 7 daysto about 21 days (such as about 7 days to about 14 days, or about 14days to about 21 days) prior to the administration of the activated Tcells. In some embodiments, the antigen presenting cells areadministered for at least three times. In some embodiments, the antigenpresenting cells are administered subcutaneously, intradermally orintravenously. In some embodiments, the activated T cells and thepopulation of antigen presenting cells are from the same individual. Insome embodiments, the activated T cells and/or the population of antigenpresenting cells are from the individual being treated. In someembodiments, the population of antigen presenting cells is a populationof DCs, B cells, or macrophages. In some embodiments, the antigenpresenting cells are DCs. In some embodiments, the MASCT furthercomprises administering to the individual an effective amount of animmune checkpoint inhibitor. In some embodiments, the activated T cellsand the immune checkpoint inhibitor are administered simultaneously,such as in the same composition. In some embodiments, the activated Tcells and the immune checkpoint inhibitor are administered sequentially.

In some embodiments, the MASCT comprises: (a) administering to theindividual an effective amount of DCs loaded with a plurality of tumorantigen peptides; (b) co-culturing a population of DCs loaded with theplurality of tumor antigen peptides and a population of T cells toobtain a population of activated T cells; and (c) administering to theindividual an effective amount of the activated T cells. In someembodiments, the interval between the administration of the DCs and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the DCs loadedwith the plurality of tumor antigen peptides are administeredsubcutaneously. In some embodiments, the DCs loaded with the pluralityof tumor antigen peptides are administered for at least three times. Insome embodiments, the activated T cells are administered intravenously.In some embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the population of T cells isco-cultured with the population of DCs loaded with the plurality oftumor antigen peptides for about 7 days to about 21 days (such as about7 days to about 10 days, about 10 days to about 15 days, about 15 daysto about 21 days, about 14 days to about 21 days, or about 10 days). Insome embodiments, the population of T cells is derived from thenon-adherent portion of a population of peripheral blood mononuclearcells (PBMCs). In some embodiments, the co-culturing further comprisescontacting the activated T cells with a plurality of cytokines (such asIL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally ananti-CD3 antibody. In some embodiments, the population of T cells iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofPD-1, PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the population of DCs loaded with the plurality of tumorantigen peptides is prepared by contacting a population of DCs with theplurality of tumor antigen peptides. In some embodiments, the populationof T cells and the population of DCs are derived from the sameindividual. In some embodiments, the population of T cells, thepopulation of DCs, the population of PBMCs, or any combination thereofis derived from the individual being treated. In some embodiments, theMASCT further comprises administering to the individual an effectiveamount of an immune checkpoint inhibitor. In some embodiments, theactivated T cells and the immune checkpoint inhibitor are administeredsimultaneously, such as in the same composition. In some embodiments,the activated T cells and the immune checkpoint inhibitor areadministered sequentially.

In some embodiments, the MASCT comprises: (a) inducing differentiationof a population of monocytes into a population of DCs; (b) contactingthe population of DCs with a plurality of tumor antigen peptides toobtain a population of DCs loaded with the plurality of tumor antigenpeptides; (c) administering to the individual an effective amount of theDCs loaded with the plurality of tumor antigen peptides; (d)co-culturing the population of DCs loaded with the plurality of tumorantigen peptides and a population of non-adherent PBMCs to obtain thepopulation of activated T cells; and (e) administering to the individualan effective amount of the activated T cells, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs. In some embodiments, the interval between theadministration of the DCs and the administration of the activated Tcells is about 7 days to about 21 days (such as about 7 days to about 14days, about 14 days to about 21 days, about 10 days or about 14 days).In some embodiments, the DCs loaded with the plurality of tumor antigenpeptides are administered subcutaneously. In some embodiments, the DCsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, or about 10 days). Insome embodiments, the co-culturing further comprises contacting theactivated T cells with a plurality of cytokines (such as IL-2, IL-7,IL-15, IL-21, or any combination thereof) and optionally an anti-CD3antibody. In some embodiments, the population of non-adherent PBMCs iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofPD-1, PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the population of PBMCs is obtained from the individualbeing treated. In some embodiments, the MASCT further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor. In some embodiments, the activated T cells and theimmune checkpoint inhibitor are administered simultaneously, such as inthe same composition. In some embodiments, the activated T cells and theimmune checkpoint inhibitor are administered sequentially.

In some embodiments, the MASCT comprises: contacting a population ofperipheral blood mononuclear cells (PBMCs) with a plurality of tumorantigen peptides to obtain a population of activated PBMCs, andadministering to the individual an effective amount of the activatedPBMCs. In some embodiments, the population of PBMCs is contacted withthe plurality of tumor antigen peptides in the presence of a compositionthat facilitates the uptake of the plurality of tumor antigen peptidesby antigen presenting cells (such as DCs) in the PBMCs. In someembodiments, the population of PBMCs is contacted with the plurality oftumor antigen peptides in the presence of an immune checkpointinhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3,BTLA, VISTA, and LAG-3. In some embodiments, the population of activatedPBMCs is contacted with IL-2. In some embodiments, the activated PBMCsare administered for at least three times. In some embodiments, theinterval between each administration of the activated PBMCs is about 2weeks to about 5 months (such as about 3 months). In some embodiments,the activated PBMCs are administered intravenously. In some embodiments,the population of PBMCs is obtained from the individual being treated.In some embodiments, the MASCT further comprises administering to theindividual an effective amount of an immune checkpoint inhibitor. Insome embodiments, the activated T cells and the immune checkpointinhibitor are administered simultaneously, such as in the samecomposition. In some embodiments, the activated T cells and the immunecheckpoint inhibitor are administered sequentially.

In some embodiments, the MASCT comprises: (a) co-culturing a populationof DCs loaded with a plurality of tumor antigen peptides and apopulation of T cells in an initial co-culture medium comprising one ormore cytokines (e.g., a plurality of cytokines) and an immune checkpointinhibitor to provide a co-culture; b) adding an anti-CD3 antibody to theco-culture at about 3 to 7 days after the co-culturing starts, therebyobtaining the population of activated T cells; and (c) administering tothe individual an effective amount of the activated T cells. In someembodiments, the plurality of cytokines comprises IL-2, IL-7, IL-15 andIL-21. In some embodiments, the IL-2 is present in the initialco-culture medium at a concentration of at least about 500 IU/mL. Insome embodiments, the immune checkpoint inhibitor is an anti-PD-1antibody. In some embodiments, the anti-PD-1 antibody is present in theinitial co-culture medium at a concentration of at least about 10 µg/mL.In some embodiments, the anti-CD3 antibody is added to the co-culture atabout 5 days after the co-culturing starts. In some embodiments, thepopulation of DCs loaded with the plurality of tumor antigen peptidesand the population of T cells are co-cultured for at least about 10 daysin the presence of the anti-CD3 antibody. In some embodiments, thepopulation of T cells is present in a population of PBMCs. In someembodiments, the population of DCs and the population of T cells areobtained from the individual being treated. In some embodiments, theactivated T cells are administered to the individual for at least threetimes. In some embodiments, the activated T cells are administeredintravenously. In some embodiments, the method further comprisesadministering to the individual an effective amount of DCs loaded withthe plurality of tumor antigen peptides. In some embodiments, the DCsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the DCs loaded with theplurality of tumor antigen peptides are administered subcutaneously,intradermally or intravenously.

In some embodiments, the MASCT comprises: a) contacting a population ofDCs with a plurality of tumor antigen peptides to obtain a population ofDCs loaded with the plurality of tumor antigen peptides; b) culturingthe population of DCs loaded with the plurality of tumor antigenpeptides in a DC maturation medium comprising MPLA; c) co-culturing thepopulation of DCs loaded with the plurality of tumor antigen peptidesand a population of T cells, thereby obtaining the population ofactivated T cells; and d) administering to the individual an effectiveamount of the activated T cells. In some embodiments, step c) comprisesco-culturing the population of DCs loaded with the plurality of tumorantigen peptides and a population of T cells in a co-culture mediumcomprising an interleukin cocktail, an immune checkpoint inhibitor andan anti-CD3 antibody. In some embodiments, the population of DCs loadedwith the plurality of tumor antigen peptides and the population of Tcells are co-cultured for at least about 10 days in the presence of theanti-CD3 antibody. In some embodiments, the DC maturation mediumcomprises INFy and MPLA. In some embodiments, the DC maturation mediumfurther comprises PGE2. In some embodiments, the MPLA is present in theDC maturation medium at a concentration of at least about 0.5 µg/mL. Insome embodiments, the INFy is present in the DC maturation medium at aconcentration of at least about 100 IU/mL. In some embodiments, the PGE2is present in the DC maturation medium at a concentration of at leastabout 0.1 µg/mL. In some embodiments, the plurality of cytokinescomprises IL-2, IL-7, IL-15 and IL-21. In some embodiments, the IL-2 ispresent in the co-culture medium at a concentration of at least about500 IU/mL. In some embodiments, the immune checkpoint inhibitor is ananti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody ispresent in the co-culture medium at a concentration of at least about 10µg/mL. In some embodiments, the population of DCs loaded with theplurality of tumor antigen peptides and the population of T cells areco-cultured for at least about 10 days in the presence of the anti-CD3antibody. In some embodiments, the population of T cells is present in apopulation of PBMCs. n some embodiments, the population of DCs and thepopulation of T cells are obtained from the individual being treated. Insome embodiments, the activated T cells are administered to theindividual for at least three times. In some embodiments, the activatedT cells are administered intravenously. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofDCs loaded with the plurality of tumor antigen peptides. In someembodiments, the DCs loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, the DCsloaded with the plurality of tumor antigen peptides are administeredsubcutaneously, intradermally or intravenously.

In some embodiments, the MASCT comprises: a) contacting a population ofDCs with a plurality of tumor antigen peptides to obtain a population ofDCs loaded with the plurality of tumor antigen peptides; b) co-culturingthe population of DCs loaded with the plurality of tumor antigenpeptides and a population of T cells in an initial co-culture mediumcomprising one or more cytokines (e.g., a plurality of cytokines) and animmune checkpoint inhibitor to provide a co-culture; c) adding ananti-CD3 antibody to the co-culture at about 3 to 7 days after theco-culturing starts, thereby obtaining the population of activated Tcells; and d) administering to the individual an effective amount of theactivated T cells. In some embodiments, step (a) further comprisesculturing the population of DCs loaded with the plurality of tumorantigen peptides in a DC maturation medium comprising a toll-likereceptor (TLR) agonist. In some embodiments, the TLR agonist is selectedfrom the group consisting of MPLA, Poly I:C, resquimod, gardiquimod, andCL075. In some embodiments, the DC maturation medium comprises PGE2. Insome embodiments, the plurality of cytokines comprises IL-2, IL-7, IL-15and IL-21. In some embodiments, the IL-2 is present in the initialco-culture medium at a concentration of at least about 500 IU/mL. Insome embodiments, the immune checkpoint inhibitor is an anti-PD-1antibody. In some embodiments, the anti-PD-1 antibody is present in theinitial co-culture medium at a concentration of at least about 10 µg/mL.In some embodiments, the anti-CD3 antibody is added to the co-culture atabout 5 days after the co-culturing starts. In some embodiments, thepopulation of DCs loaded with the plurality of tumor antigen peptidesand the population of T cells are co-cultured for at least about 10 daysin the presence of the anti-CD3 antibody. In some embodiments, thepopulation of DCs and the population of T cells are obtained from theindividual being treated. In some embodiments, the activated T cells areadministered to the individual for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the method further comprises administering to theindividual an effective amount of DCs loaded with the plurality of tumorantigen peptides. In some embodiments, the DCs loaded with the pluralityof tumor antigen peptides are administered for at least three times. Insome embodiments, the DCs loaded with the plurality of tumor antigenpeptides are administered subcutaneously, intradermally orintravenously.

In some embodiments, the MASCT comprises: a) contacting a population ofDCs with a plurality of tumor antigen peptides to obtain a population ofDCs loaded with the plurality of tumor antigen peptides; b) culturingthe population of DCs loaded with the plurality of tumor antigenpeptides in a DC maturation medium comprising MPLA; c) co-culturing thepopulation of DCs loaded with the plurality of tumor antigen peptidesand a population of T cells in an initial co-culture medium comprisingone or more cytokines (e.g., a plurality of cytokines) and an immunecheckpoint inhibitor to provide a co-culture; d) adding an anti-CD3antibody to the co-culture, thereby obtaining the population ofactivated T cells; and e) administering to the individual an effectiveamount of the activated T cells. In some embodiments, the anti-CD3antibody is added to the co-culture when the co-culturing starts. Insome embodiments, the anti-CD3 antibody is added to the co-culture afterthe co-culturing starts. In some embodiments, the DC maturation mediumcomprises INFy and MPLA. In some embodiments, the DC maturation mediumfurther comprises PGE2. In some embodiments, the MPLA is present in theDC maturation medium at a concentration of at least about 0.5 µg/mL. Insome embodiments, the INFy is present in the DC maturation medium at aconcentration of at least about 100 IU/mL. In some embodiments, the PGE2is present in the DC maturation medium at a concentration of at leastabout 0.1 µg/mL. In some embodiments, the plurality of cytokinescomprises IL-2, IL-7, IL-15 and IL-21. In some embodiments, the IL-2 ispresent in the initial co-culture medium at a concentration of at leastabout 500 IU/mL. In some embodiments, the immune checkpoint inhibitor isan anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody ispresent in the initial co-culture medium at a concentration of at leastabout 10 µg/mL. In some embodiments, the population of DCs loaded withthe plurality of tumor antigen peptides and the population of T cellsare co-cultured for at least about 10 days in the presence of theanti-CD3 antibody. In some embodiments, the population of DCs and thepopulation of T cells are obtained from the individual being treated. Insome embodiments, the activated T cells are administered to theindividual for at least three times. In some embodiments, the activatedT cells are administered intravenously. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofDCs loaded with the plurality of tumor antigen peptides. In someembodiments, the DCs loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, the DCsloaded with the plurality of tumor antigen peptides are administeredsubcutaneously, intradermally or intravenously.

In some embodiments, the MASCT comprises: a) contacting a population ofDCs with a plurality of tumor antigen peptides to obtain a population ofDCs loaded with the plurality of tumor antigen peptides; b) culturingthe population of DCs loaded with the plurality of tumor antigenpeptides in a DC maturation medium comprising MPLA, INFy and PGE2; c)co-culturing the population of DCs loaded with the plurality of tumorantigen peptides and a population of T cells in an initial co-culturemedium comprising a plurality of cytokines comprising IL-2, IL-7, IL-15and IL-21 and an anti-PD-1 antibody to provide a co-culture; d) addingan anti-CD3 antibody to the co-culture at about 3 to 7 days (e.g., about5 days) after the co-culturing starts, thereby obtaining the populationof activated T cells; and e) administering to the individual aneffective amount of the activated T cells. In some embodiments, the MPLAis present in the DC maturation medium at a concentration of at leastabout 0.5 µg/mL. In some embodiments, the INFγ is present in the DCmaturation medium at a concentration of at least about 100 IU/mL. Insome embodiments, the PGE2 is present in the DC maturation medium at aconcentration of at least about 0.1 µg/mL. In some embodiments, the IL-2is present in the initial co-culture medium at a concentration of atleast about 500 IU/mL. In some embodiments, the anti-PD-1 antibody ispresent in the initial co-culture medium at a concentration of at leastabout 10 µg/mL. In some embodiments, the population of DCs loaded withthe plurality of tumor antigen peptides and the population of T cellsare co-cultured for at least about 10 days in the presence of theanti-CD3 antibody. In some embodiments, the population of DCs and thepopulation of T cells are obtained from the individual being treated. Insome embodiments, the activated T cells are administered to theindividual for at least three times. In some embodiments, the activatedT cells are administered intravenously. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofDCs loaded with the plurality of tumor antigen peptides. In someembodiments, the DCs loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, the DCsloaded with the plurality of tumor antigen peptides are administeredsubcutaneously, intradermally or intravenously.

Generally, dosages, schedules, and routes of administration of theactivated T cells and the population of DCs loaded with the plurality oftumor antigen peptides described herein may be determined according tothe size and condition of the individual, and according to standardpharmaceutical practice. Exemplary routes of administration includeintravenous, intra-arterial, intraperitoneal, intrapulmonary,intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, or transdermal. In some embodiments, the DCsloaded with the plurality of tumor antigen peptides are administeredsubcutaneously. In some embodiments, the activated T cells areadministered intravenously.

The dose of the cells administered to an individual may vary accordingto, for example, the particular type of cells being administered, theroute of administration, and the particular type and stage of cancerbeing treated. The amount should be sufficient to produce a desirableresponse, such as a therapeutic response against cancer, but withoutsevere toxicity or adverse events. In some embodiments, the amount ofthe activated T cells or the DCs to be administered is a therapeuticallyeffective amount. In some embodiments, the amount of the cells (such asmultiple-antigen loaded DCs, or the activated T cells) is an amountsufficient to decrease the size of a tumor, decrease the number ofcancer cells, or decrease the growth rate of a tumor by at least aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% comparedto the corresponding tumor size, number of cancer cells, or tumor growthrate in the same individual prior to treatment or compared to thecorresponding activity in other individuals not receiving the treatment.Standard methods can be used to measure the magnitude of this effect,such as in vitro assays with purified enzyme, cell-based assays, animalmodels, or human testing.

In some embodiments, the antigen-loaded dendritic cells are administeredat a dose at least about any one of 1x10⁵, 5x10⁵, 1x10⁶, 1.5x10⁶, 2x10⁶,3x10⁶, 4x10⁶, 5x10⁶, 6x10⁶, 7x10⁶, 8x10⁶, 9x10⁶, 1x10⁷ or 5 x10⁷cells/individual. In some embodiments, the antigen-loaded dendriticcells are administered at a dose about any one of 1x10⁵-5x10⁵,5x10⁵-1x10⁶, 1x10⁶-2x10⁶ 2x10⁶-3x10⁶, 3x10⁶-4x10⁶ 4x10⁶-5x10⁶5x10⁶-6x10⁶ 6x10⁶ -7x10⁶ 7x10⁶-8x10⁶, 8x10⁶-1x10⁸ , 1x10⁶-3x10⁶ ,3x10⁶-5x10^(6 >) 5x10⁶-7x10⁶ , 2x10⁶-2x10⁷ , 5x10⁶-2x10⁷, or 1x10⁶-2x10⁷ cells/individual. In some embodiments, the antigen-loaded dendriticcells are administered at a dose of at least about 1 x 10⁶cells/individual. In some embodiments, the antigen-loaded dendriticcells are administered at a dose of about 1.5x10⁶ to about 1.5 x10⁷cells/ individual.

In some embodiments, the antigen-loaded dendritic cells are administeredat a dose at least about any one of 1x10⁴, 2.5x10⁴, 5x10⁴, 1x10⁵, 2x10⁵,2.5x10⁵, 4x10⁵, 6x10⁵, 8x10⁵, 1x10⁶, 2x10⁶ or 1x10⁷ cells/kg. In someembodiments, the antigen-loaded dendritic cells are administered at adose about any one of 1x10⁴-5x10⁴, 5x10⁴-1x10⁵, 1x10⁵-2x10⁵, 2x10⁵-4x10⁵4x10⁵-6x10⁵ 6x10⁵-8x10⁵ 8x10⁵-1x10⁶ 1x10⁶-2x10⁶ 2x10⁶-1x10^(7,)1x10⁴-1x10⁵ , 1x10⁵-1x10⁶, 1x10⁶-1x10⁷, 1x10⁴-1x10⁶, or 1x10⁵-1 x10⁷cells/kg. In some embodiments, the antigen-loaded dendritic cells areadministered at a dose of at least about 2 x 10⁵ cells/kg. In someembodiments, the antigen-loaded dendritic cells are administered at adose of about 2.5x10⁴ to about 2.5 x10⁵ cells/kg.

In some embodiments, the activated T cells are administered at a dose ofat least about any one of 1 x 10⁸, 5 x 10⁸, 1 x 10⁹, 2 x 10⁹, 3x10⁹, 4 x10⁹, 5 x 10⁹, 6 x 10⁹, 7 x 10⁹, 8 x 10⁹, 9 x 10⁹, 1 x10¹⁰, 1.5 x 10¹⁰, 2x10¹⁰, or 5 x 10¹⁰ cells/individual. In some embodiments, the activatedT cells are administered at a dose of about any one of 1 x 10⁸-5 x10⁸- x10⁸-1 x10⁹, 1 x 10⁹-5 x 10⁹, 5 x 10⁹-1 x 10¹⁰, 3 x 10⁹-7 x 10⁹, 1 x10¹⁰-2 x 10¹⁰, or 1 x 10⁹-1 x 10¹⁰ cells/individual. In someembodiments, the activated T cells are administered at a dose of atleast about 3 x 10⁹ cells/individual. In some embodiments, the activatedT cells are administered at a dose of about 1 x 10⁹ to about 1 x 10¹⁰cells/individual.

In some embodiments, the activated T cells are administered at a dose ofat least about any one of 1 x 10⁷, 2 x 10⁷, 4 x 10⁷, 6x10⁷, 8 x 10⁷, 1 x10⁸, 2 x 10⁸ 4 x 10⁸, 6 x 10⁸, 8 x 10⁸, 1 x10⁹ cells/kg. In someembodiments, the activated T cells are administered at a dose of aboutany one of 1 x 10⁷-1 x 10⁸, 1 x 10⁷-5 x10⁷, 2 x 10⁷-4 x10⁷, 5 x 10⁷-1 x10⁸, 1 x 10⁸-2 x 10⁸, 5 x 10⁷-1 x 10⁸, 1 x 10⁸-2 x 10⁸, 2x 10⁸-5 x 10⁸,1 x 10⁸-1 x 10⁹, or 1 x 10⁷ - 1 x 10⁹ cells/kg. In some embodiments, theactivated T cells are administered at a dose of at least about 6 x 10⁷cells/kg. In some embodiments, the activated T cells are administered ata dose of about 1.5 x 10⁷ to about 2 x 10⁸ cells/kg.

The MASCT can be used in monotherapy as well as in combination therapywith another agent. For example, any of the treatment methods describedherein may be combined with administration of one or more (such as anyof 1, 2, 3, 4, or more) immune checkpoint inhibitors. In someembodiments, the immune checkpoint inhibitor is selected from the groupconsisting of inhibitors of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA,VISTA, and LAG-3.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-1. In some embodiments, the immune checkpoint inhibitor is ananti-PD-1 antibody. Exemplary anti-PD-1 antibodies include, but are notlimited to, Nivolumab, pembrolizumab, pidilizumab, BMS-936559, andatezolizumab, Pembrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, andTSR-042. In some embodiments, the immune checkpoint inhibitor isnivolumab (for example, OPDIVO®). In some embodiments, the immunecheckpoint inhibitor is Pembrolizumab (for example, KEYTRUDA®). In someembodiments, the immune checkpoint inhibitor is SHR-1210.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is ananti-PD-L1 antibody. Exemplary anti-PD-L1 antibodies include, but arenot limited to, KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A,MEDI4736, Avelumab, or STI-A1010.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofCTLA-4. In some embodiments, the immune checkpoint inhibitor is ananti-CTLA-4 antibody. Exemplary anti-CTLA-4 antibodies include, but arenot limited to, Ipilimumab, Tremelimumab, and KAHR-102. In someembodiments, the immune checkpoint inhibitor is Ipilimumab (for example,YERVOY®).

In some embodiments, the activated T cells and the immune checkpointinhibitor are administered simultaneously. In some embodiments, theactivated T cells and the immune checkpoint inhibitor are administeredin a single composition. In some embodiments, the immune checkpointinhibitor is present in the first, second or third co-culture. In someembodiments, the activated T cells and the immune checkpoint inhibitorare admixed prior to (such as immediately prior to) the administration.In some embodiments, the activated T cells and the immune checkpointinhibitor are administered simultaneously via separate compositions.

In some embodiments, the activated T cells and the immune checkpointinhibitor are administered sequentially. In some embodiments, the immunecheckpoint inhibitor is administered prior to the administration of theactivated T cells. In some embodiments, the immune checkpoint inhibitoris administered after the administration of the activated T cells.

Exemplary routes of administration of the immune checkpoint inhibitorinclude, but are not limited to, intratumoral, intravesical,intramuscular, intraperitoneal, intravenous, intra-arterial,intracranial, intrapleural, subcutaneous, and epidermal routes, or bedelivered into lymph glands, body spaces, organs or tissues known tocontain such live cancer cells. In some embodiments, the immunecheckpoint inhibitor is administered intravenously. In some embodiments,the immune checkpoint inhibitor is administered by infusion. In someembodiments, the immune checkpoint inhibitor is infused over at leastabout any of 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours,or more. In some embodiments, the immune checkpoint inhibitor isadministered via the same administration route as the activated T cells.In some embodiments, the immune checkpoint inhibitor is administered viaa different administration route as the activated T cells.

Suitable dose of the immune checkpoint inhibitor include, but are notlimited to, about any one of 1 mg/m², 5 mg/m², 10 mg/m², 20 mg/m², 50mg/m², 100 mg/m², 200 mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 750 mg/m²,1000 mg/m², or more. In some embodiments, the dose of immune checkpointinhibitor is any one of about 1 to about 5 mg/m², about 5 to about 10mg/m², about 10 to about 20 mg/m², about 20 to about 50 mg/m², about 50to about 100 mg/m², about 100 mg/m² to about 200 mg/m², about 200 toabout 300 mg/m², about 300 to about 400 mg/m², about 400 to about 500mg/m², about 500 to about 750 mg/m², or about 750 to about 1000 mg/m².In some embodiments, the dose of immune checkpoint inhibitor is aboutany one of 1 µg/kg, 2 µg/kg, 5 µg/kg, 10 µg/kg, 20 µg/kg, 50 µg/kg, 0.1mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, or more. In someembodiments, the dose of the immune checkpoint inhibitor is any one ofabout 1 µg/kg to about 5 µg/kg, about 5 µg/kg to about 10 µg/kg, about10 µg/kg to about 50 µg/kg, about 50 µg/kg to about 0.1 mg/kg, about 0.1mg/kg to about 0.2 mg/kg, about 0.2 mg/kg to about 0.3 mg/kg, about 0.3mg/kg to about 0.4 mg/kg, about 0.4 mg/kg to about 0.5 mg/kg, about 0.5mg/kg to about 1 mg/kg, about 1 mg/kg to about 5 mg/kg, about 5 mg/kg toabout 10 mg/kg, about 10 mg/kg to about 20 mg/kg, about 20 mg/kg toabout 50 mg/kg, about 50 mg/kg to about 100 mg/kg, or about 1 mg/kg toabout 100 mg/kg.

In some embodiments, the immune checkpoint inhibitor is administereddaily. In some embodiments, the immune checkpoint inhibitor isadministered is administered at least about any one of 1x, 2x, 3x, 4x,5x, 6x, or 7x (i.e., daily) a week. In some embodiments, the immunecheckpoint inhibitor is administered weekly. In some embodiments, theimmune checkpoint inhibitor is administered weekly without break;weekly, two out of three weeks; weekly three out of four weeks; onceevery two weeks; once every 3 weeks; once every 4 weeks; once every 6weeks; once every 8 weeks, monthly, or every two to 12 months. In someembodiments, the intervals between each administration are less thanabout any one of 6 months, 3 months, 1 month, 20 days, 15 days, 12 days,10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days,or 1 day. In some embodiments, the intervals between each administrationare more than about any one of 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 8 months, or 12 months. In some embodiments, theimmune checkpoint inhibitor is administered once every 3 months. In someembodiments, there is no break in the dosing schedule. In someembodiments, the interval between each administration is no more thanabout a week. In some embodiments, the immune checkpoint inhibitor isadministered with the same dosing schedule as the activated T cells. Insome embodiments, the immune checkpoint inhibitor is administered with adifferent dosing schedule as the activated T cells.

In some embodiments, the immune checkpoint inhibitor is administered inevery MASCT treatment cycle. For example, the immune checkpointinhibitor may be administered about any of 1, 2, 3, 4, 5, 6, or moretimes every MASCT treatment cycle. In some embodiments, the immunecheckpoint inhibitor is not administered in every MASCT treatment cycle.For example, the immune checkpoint inhibitor may be administered aboutonce every 1, 2, 3, 4, 5, or more MASCT treatment cycles.

The administration of the immune checkpoint inhibitor can be over anextended period of time, such as from about a month up to about sevenyears. In some embodiments, the immune checkpoint inhibitor isadministered over a period of at least about any one of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. Insome embodiments, the immune checkpoint inhibitor is administered for asingle time. In some embodiments, the immune checkpoint inhibitor isadministered repeatedly. In some embodiments, the immune checkpointinhibitor is administered repeatedly until disease progression.

In some embodiments, the MASCT is particularly suitable for anindividual with a low total mutation load in the cancer of theindividual. In some embodiments, the MASCT is particularly suitable foran individual with a low mutation load in the cancer-associated genes inthe cancer of the individual. In some embodiments, the MASCT isparticularly suitable for an individual with a low mutation load inimmune genes related to T cell response in the cancer of the individual.In some embodiments, the MASCT is particularly suitable for anindividual with a low mutation load in the MHC genes in the cancer ofthe individual. The mutation load may be mutation load in all cancercells, or a subset of cancer cells, such as a primary or metastatictumor site, for example, cells in a tumor biopsy sample.

In some embodiments, a low mutation load of one or more genes is a lownumber of mutations accumulated on the one or more genes. In someembodiments, a total number of no more than about any of 500, 400, 300,200, 100, 50, 40, 30, 20, 10, 5 or fewer mutations indicate a lowmutation load. In some embodiments, no more than about any of 50, 40,30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1mutations in the one or more MHC genes indicate a low mutation load ofthe one or more MHC genes. In some embodiments, a low mutation load ofone or more genes is a low ratio between the number of mutationsaccumulated on the one or more genes (such as MHC genes) and the totalnumber of mutations in a selected set of genes (such ascancer-associated genes) or the full genome.

In some embodiments, the one or more MHC genes comprise MHC class Igenes (or loci). In some embodiments, the one or more MHC genes compriseMHC class II genes (or loci). In some embodiments, wherein theindividual is a human individual, the one or more MHC genes are selectedfrom the group consisting of HLA-A, HLA-B, HLA-C and B2M.

Exemplary mutations include, but are not limited to, deletion,frameshift, insertion, indel, missense mutation, nonsense mutation,point mutation, copy number variation, single nucleotide variation(SNV), silent mutation, splice site mutation, splice variant, genefusion, and translocation. In some embodiments, the copy numbervariation of the MHC gene is caused by structural rearrangement of thegenome, including deletions, duplications, inversion, and translocationof a chromosome or a fragment thereof. In some embodiments, themutations in the one or more MHC genes are selected from pointmutations, frameshift mutations, gene fusions, and copy numbervariations. In some embodiments, the mutations are in the protein-codingregion of the MHC genes. In some embodiments, the mutation is anonsynonymous mutation. In some embodiments, the mutation is not apolymorphism. In some embodiments, the mutation is present in normalcells of the individual. In some embodiments, the mutation is notpresent in normal cells of the individual. In some embodiments, themutation affects the physiochemical or functional properties, such asstability or binding affinity, of the MHC molecule encoded by theaffected gene. In some embodiments, the mutation results in anirreversible deficiency in the MHC molecule. In some embodiments, themutation reduces the binding affinity of the MHC molecule to T cellepitopes and/or T cell receptors. In some embodiments, the mutation is aloss-of-function mutation. In some embodiments, the mutation results inreversible deficiency in the MHC molecule. In some embodiments, themutation does not affect the binding affinity of the MHC molecule to Tcell epitopes and/or T cell receptors. In some embodiments, the mutationis a somatic mutation. In some embodiments, the mutation is a germlinemutation.

The mutations counted towards the mutation load may be present in allcancer cells or in a subset of cancer cells. In some embodiments, themutations are present in all cancer cells in the individual. In someembodiments, the mutations are present in all cancer cells of a tumorsite. In some embodiments, the mutations are clonal. In someembodiments, the mutations are subclonal. In some embodiments, themutations are present in at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or more cancer cells of the individual.

The mutations in certain MHC genes and/or in certain domains orpositions of the one or more MHC genes may have more profound influenceon the clinical response of the individual to the treatment methodsdescribed herein. For example, loss-of-function mutations may occur inthe leader peptide sequence, a3 domain (which binds the CD8 co-receptorof T cells), a1 peptide binding domain, or a2 peptide binding domain ofthe HLA molecule; see, for example, Shukla S. et al. NatureBiotechnology 33, 1152-1158 (2015), incorporated herein by reference.Mutations in B2M (β2-macroglobulin) gene may also promote tumor escapephenotypes. See, for example, Monica B et al. Cancer Immunol. Immu.,(2012) 61: 1359-1371. In some embodiments, presence of any number (suchas 1, 2, 3, 4, 5, or more) of mutations in the functional regions of theone or more MHC genes, such as the leader peptide sequence, a1 domain,a2 domain, or a3 domain, indicates a high mutation load. In someembodiments, presence of any number (such as 1, 2, 3, 4, 5, or more)loss-of-function mutations in the one or more MHC genes (such as HLA-A,HLA-B or HLA-C genes in human individuals) indicates a high mutationload. In some embodiments, a low mutation load in the one or more MHCgenes comprises no mutation in the functional regions, including leaderpeptide sequence, a1 domain (for example, residues in direct contactwith the CD8 co-receptor), a2 domain, and a3 domain (for example,residues in direct contact with the epitope), of the one or more MHCgenes (such as HLA-A, HLA-B or HLA-C genes). In some embodiments,presence of any number of mutations (such as loss-of-function mutations)in the B2M gene indicates a high mutation load. In some embodiments, alow mutation load in the one or more MHC genes comprises no mutation inthe B2M gene.

The mutation load of one or more genes (such as MHC genes) may bedetermined by any known methods in the art, including, but not limitedto, genomic DNA sequencing, exome sequencing, or other DNAsequencing-based methods using Sanger sequencing or next generationsequencing platforms; polymerase chain reaction assays; in situhybridization assays; and DNA microarrays.

In some embodiments, the mutation load of the one or more MHC genes isdetermined by sequencing a tumor sample from the individual. In someembodiments, the sequencing is next generation sequencing. In someembodiments, the sequencing is full genome sequencing. In someembodiments, the sequencing is exome sequencing, such as whole exomesequencing (“WES”). In some embodiments, the sequencing is RNAsequencing. In some embodiments, the sequencing is targeted sequencingof candidate genes, such as cancer-associated genes plus HLA genes. Forexample, ONCOGXONE™ Plus (Admera Health), are available to sequencecancer-associated genes and HLA loci with high sequencing depth. In someembodiments, the same sequencing data can be used to determine themutation load of the one or more MHC genes and to identify neoantigensin the individual.

In some embodiments, the tumor sample is a tissue sample. In someembodiments, the tumor sample is a tumor biopsy sample, such as fineneedle aspiration of tumor cells or laparoscopy obtained tumor cells(such as including tumor stroma). In some embodiments, the tumor sampleis freshly obtained. In some embodiments, the tumor sample is frozen. Insome embodiments, the tumor sample is a Formaldehyde Fixed-ParaffinEmbedded (FFPE) sample. In some embodiments, the tumor sample is a cellsample. In some embodiments, the tumor sample comprises a circulatingmetastatic cancer cell. In some embodiments, the tumor sample isobtained by sorting circulating tumor cells (CTCs) from blood. In someembodiments, nucleic acids (such as DNA and/or RNA) are extracted fromthe tumor sample for the sequencing analysis. In some embodiments, thesequencing data of the tumor sample is compared to the sequencing dataof a reference sample, such as a sample of a healthy tissue from thesame individual, or a sample of a healthy individual, to identifymutations and determine mutation load in the tumor cells. In someembodiments, the sequencing data of the tumor sample is compared to thereference sequences from a genome database to identify mutations anddetermine mutation load in the tumor cells.

Any of the MASCT methods may comprise using one or more neoantigenpeptides in the plurality of tumor antigen peptides. In someembodiments, the MASCT further comprises the steps of selecting theindividual for the method of treating based on having one or more (suchas at least 5) neoantigens in the individual, and/or the steps of: (i)identifying a neoantigen of the individual; and (ii) incorporating aneoantigen peptide derived from the neoantigen in the plurality of tumorantigen peptides for use in the treatment method.

In some embodiments, the MASCT comprises: (a) identifying a neoantigenof the individual; (b) incorporating a neoantigen peptide in a pluralityof tumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen; (c) optionally administering an effectiveamount of DCs loaded with the plurality of tumor antigen peptides; (d)preparing a population of activated T cells by co-culturing theantigen-loaded DCs with a population of T cells; and (e) administeringto the individual an effective amount of activated T cells, wherein theindividual has one or more neoantigens.

The individual may have any number (such as at least about any one of 1,2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 100 or more) ofneoantigens in order to benefit from the MASCT method using a pluralityof tumor antigen peptides comprising a neoantigen peptide. In someembodiments, the MASCT method is particularly suitable for an individualhaving at least about any one of 4, 5, 6, 7, 8, 10, 15, 20, 50, 100 ormore neoantigens. In some embodiments, the neoantigen comprises one ormore neoepitopes. In some embodiments, the MASCT method is particularlysuitable for an individual having at least about any one of 4, 5, 6, 7,8, 10, 15, 20, 50, 100 or more neoepitopes. In some embodiments, the Tcell epitopes are MHC-I restricted epitopes. In some embodiments, theneoepitope has a higher affinity to the MHC molecules of the individualthan the corresponding wildtype T cell epitope. In some embodiments, theneoepitope has higher affinity to a model T cell receptor than thecorresponding wildtype T cell epitope. In some embodiments, theneoantigen (or neoepitope) is a clonal neoantigen. In some embodiments,the neoantigen (or neoepitope) is a subclonal neoantigen. In someembodiments, the neoantigen (or neoepitope) is present in at least aboutany one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or moretumor cells in the individual.

The number of neoantigens may be combined with other biomarkers orselection criteria to select an individual for any one of the MASCTmethods described herein. In some embodiments, the MASCT method isparticularly suitable for an individual with a low mutation load (suchas in one or more MHC genes) in the cancer cells, and/or have at leastabout any of 4, 5, 6, 7, 8, 10 or more neoantigens (such as neoantigenswith high affinity MHC-I restricted neoepitopes).

Any number (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) ofneoantigen peptides may be designed based on the neoantigens of theindividual and to be incorporated in the plurality of tumor antigenpeptides for use in any of the treatment methods described herein. Insome embodiments, the plurality of tumor antigen peptides comprises asingle neoantigen peptide. In some embodiments, the plurality of tumorantigen peptides comprises a plurality of neoantigen peptides. Eachneoantigen peptide may comprise one or more neoepitopes from aneoantigen of the individual. In some embodiments, the neoepitope is a Tcell epitope. Methods of designing a neoantigen peptide based on aneoantigen are described in the section “Plurality of tumor antigenpeptides.”

The neoantigens in the individual may be identified using any knownmethods in the art. In some embodiments, the neoantigen is identifiedbased on the genetic profile of a tumor sample from the individual. Eachneoantigen comprises one or more neoepitopes. In some embodiments, theone or more neoepitopes in the neoantigen are identified based on thegenetic profile of the tumor sample. Any known genetic profilingmethods, such as next generation sequencing (NGS) methods, microarrays,or proteomic methods may be used to provide the genetic profile of thetumor sample.

In some embodiments, the neoantigen is identified by sequencing a tumorsample from the individual. In some embodiments, the sequencing is nextgeneration sequencing. In some embodiments, the sequencing isfull-genome sequencing. In some embodiments, the sequencing is exomesequencing, such as whole exome sequencing (“WES”). In some embodiments,the sequencing is RNA sequencing. In some embodiments, the sequencing istargeted sequencing of candidate genes, such as cancer-associated genes.Many commercial NGS cancer panels, for example, ONCOGXONE™ Plus (AdmeraHealth), are available to sequence cancer-associated genes with highsequencing depth.

In some embodiments, the tumor sample is a tissue sample. In someembodiments, the tumor sample is a tumor biopsy sample, such as fineneedle aspiration of tumor cells or laparoscopy obtained tumor cells(such as including tumor stroma). In some embodiments, the tumor sampleis freshly obtained. In some embodiments, the tumor sample is frozen. Insome embodiments, the tumor sample is a Formaldehyde Fixed-ParaffinEmbedded (FFPE) sample. In some embodiments, the tumor sample is a cellsample. In some embodiments, the tumor sample comprises a circulatingmetastatic cancer cell. In some embodiments, the tumor sample isobtained by sorting circulating tumor cells (CTCs) from blood. In someembodiments, nucleic acids (such as DNA and/or RNA) are extracted fromthe tumor sample for the sequencing analysis. In some embodiments,proteins are extracted from the tumor sample for the sequencinganalysis.

In some embodiments, the genetic profile of the tumor sample is comparedto the genetic profile of a reference sample, such as a sample of ahealthy tissue from the same individual, or a sample of a healthyindividual, to identify candidate mutant genes in the tumor cells. Insome embodiments, the genetic profile of the tumor sample is compared tothe reference sequences from a genome database to identify candidatemutant genes in the tumor cells. In some embodiments, the candidatemutant genes are cancer-associated genes. In some embodiments, eachcandidate mutant gene comprises one or more mutations, such asnonsynonymous substitutions, indel (insertion or deletion), or genefusion, which may give rise to a neoantigen. Common Single NucleotidePolymorphisms (SNPs) are excluded from the candidate mutations.

In some embodiments, neoepitopes in neoantigens are identified from thecandidate mutant proteins. In some embodiments, the neoepitopes arepredicted in silico. Exemplary bioinformatics tools for T cell epitopeprediction are known in the art, for example, see Yang X. and Yu X.(2009) “An introduction to epitope prediction methods and software” Rev.Med. Virol. 19(2): 77-96. Factors considered in the T cell epitopeprediction algorithms include, but are not limited to, MHC subtype ofthe individual, sequence-derived physiochemical properties of the T cellepitope, MHC binding motifs, proteasomal cleavage pattern, transporterassociated with antigen processing (TAP) transport efficiency, MHCbinding affinity, peptide-MHC stability, and T-cell receptor bindingaffinity. In some embodiments, the neoepitope is an MHC-I restrictedepitope. In some embodiments, the neoepitope is an MHC-II restrictedepitope.

In some embodiments, the neoepitope has high affinity to the MHCmolecules of the individual. In some embodiments, the method furthercomprises determining the MHC subtype of the individual, for example,from the sequencing data, to identify one or more MHC molecules of theindividual. In some embodiments, the method further comprisesdetermining the affinity of the neoepitope to an MHC molecule, such asan MHC class I molecule. In some embodiments, the method comprisesdetermining the affinity of the neoepitope to one or more MHC (such asMHC class I) molecules of the individual. In some embodiments, theaffinity of the neoepitope to one or more MHC molecules of theindividual is compared to the affinity of the corresponding wildtypeepitope to the one or more MHC molecules of the individual. In someembodiments, the neoepitope is selected for having a higher (such as atleast about any of 1.5, 2, 5, 10, 15, 20, 25, 50, 100, or more times)affinity to the one or more MHC molecules (such as MHC-I molecules) ofthe individual than the corresponding wildtype epitope. In someembodiments, the MHC binding affinity is predicted in silico using anyknown tools or methods in the art. In some embodiments, the MHC bindingaffinity is determined experimentally, such as using an in vitro bindingassay.

In some embodiments, the MASCT further comprises determining theaffinity of the complex comprising the neoepitope and an MHC molecule(such as an MHC class I molecule of the individual) to a T cellreceptor. In some embodiments, the affinity of the complex comprisingthe neoepitope and the MHC molecule to the T cell receptor is comparedto that of the complex comprising the corresponding wildtype epitope andthe MHC molecule. In some embodiments, the MHC molecule is from theindividual. In some embodiments, the T cell receptor is on the surfaceof one or more T cells of the individual. In some embodiments, theneoepitope is selected for having a higher (such as at least about anyone of 1.5, 2, 5, 10, 15, 20, 25, 50, 100, or more times) affinity in acomplex comprising the neoepitope and an MHC molecule to a T cellreceptor model than the corresponding wildtype epitope. In someembodiments, the TCR binding affinity is predicted in silico using anyknown tools or methods in the art. In some embodiments, the TCR bindingaffinity is determined experimentally, for example, by determining the Tcell response against the neoepitope.

In some embodiments, the neoantigen (or the neoepitope) is identifiedfurther based on the expression level of the neoantigen (or theneoepitope) in the tumor sample. Expression level of the neoantigen (orthe neoepitope) may be determined using any methods for quantificationof mRNA or protein levels known in the art, such as RT-PCR,antibody-based assays, mass spectrometry. In some embodiments, theexpression level of the neoantigen (or the neoepitope) is determinedfrom the sequencing data of the tumor sample. In some embodiments, theneoantigen (or the neoepitope) is expressed in the tumor cells at alevel of at least about any one of 10, 20, 50, 100, 200, 500, 1000,2000, 5000, 10⁴, or more copies per cell. In some embodiments, theneoantigen (or the neoepitope) is expressed at a level of more thanabout any one of 1.5, 2, 5, 10, 20, 50, 100, or more times than thecorresponding wildtype protein (or the corresponding wildtype epitope)in the tumor cells.

In some embodiments, the neoantigen peptide is selected or identified bythe steps comprising: (a) sequencing a tumor sample from the individualto identify a neoantigen; (b) identifying a neoepitope in theneoantigen; optionally (c) determining the MHC subtype of the individual(e.g., using the sequencing data) to identify an MHC molecule of theindividual; optionally (d) determining the affinity of the neoepitope tothe MHC molecule of the individual; optionally (e) determining theaffinity of the complex comprising the neoepitope and the MHC moleculeto a T cell receptor; and (f) obtaining a peptide comprising theneoepitope to provide the neoantigen peptide. In some embodiments, theneoepitope has higher affinity to the MHC molecule (such as MHC-Imolecule) of the individual and/or higher affinity in the complexcomprising the neoepitope and the MHC molecule to the TCR as compared tothe complex comprising the corresponding wildtype T cell epitope and theMHC molecule. In some embodiments, the neoepitope is extended at the Nterminus or the C terminus or both termini according to the naturalsequence of the neoantigen harboring the epitope to obtain an extendedsequence, wherein the extended sequence is amenable for presentation byboth class I and class II MHC molecules. Any of the treatment methodsdescribed herein using one or more neoantigen peptides may furthercomprise any one or more of the neoantigen selection/identificationsteps.

Any of the treatment methods and the MASCT methods described herein mayfurther comprise a monitoring step after the individual receives thetreatment. Post-treatment monitoring may be beneficial for adjusting thetreatment regimen of the individual to optimize treatment outcome.

For example, the plurality of tumor antigen peptides described hereinmay be adjusted or customized based on the specific immune response ofthe individual against each of the plurality of tumor antigen peptidesand/or the clinical response of the individual to the activated T cellsin order to provide a plurality of customized tumor antigen peptides,which may be used for repeated treatments. In some embodiments, tumorantigen peptides that do not elicit a strong specific immune responsecan be removed from the antigen peptide pool for future preparations ofthe pulsed DCs or activated T cells.

Specific immune response against one or more tumor antigen peptides maybe determined using any known methods in the art, for example, bymeasuring levels of cytotoxic factor (such as perforin or granzyme B),or cytokine release (such as IFNγ or TNFa) from T cells (or PBMCs) afterstimulation by the individual tumor antigen peptide. An antibody-basedassay, such as ELISPOT, may be used to quantify the cytotoxic factor, orcytokine (such as IFNy) levels. In some embodiments, the cytokine (suchas IFNy) release level from T cells (or PBMCs) in response to a tumorantigen peptide is normalized to a reference, such as a baselinecytokine release level, or a nonspecific cytokine release level of fromT cells (or PBMCs) in response to an irrelevant peptide, to provide acytokine (such as IFNy) fold change value. In some embodiments, acytokine (such as IFNy) fold change value of more than about any one of1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, or more in an ELISPOT assayindicate strong specific immune response against the tumor antigenpeptide. In some embodiments, a tumor antigen peptide with a cytokine(such as IFNy) fold change value of less than about any one of 10, 8, 6,5, 4, 3, 2.5, 2, 1.5, 1.2 or less in an ELISPOT assay is removed fromthe plurality of tumor antigen peptides to provide a plurality ofcustomized tumor antigen peptides for future treatments.

Clinical response of the individual to the treatment methods describedherein may be assessed by known methods in the art by a physician, suchas by imaging methods, blood tests, biomarker assessment, and biopsy. Insome embodiments, the clinical response is monitored by determining thenumber of circulating tumor cells (CTC) in the individual before andafter receiving the activated T cells. In some embodiments, the CTCshave detached from a primary tumor and circulate in a bodily fluid. Insome embodiments, the CTCs have detached from a primary tumor andcirculate in the bloodstream. In some embodiments, the CTCs are anindication of metastasis. CTC numbers can be determined by a variety ofmethods known in the art, including, but not limited to, CellSearchmethod, Epic Science method, isoflux, and maintrac. In some embodiments,the number of single CTCs, including specific subtypes of CTCs, in ablood sample of the individual is determined. In some embodiments, anumber of more than about any of 10, 20, 50, 100, 150, 200, 300 or moreof single CTCs per mL of the blood sample in the individual afterreceiving the treatment indicates an increased risk of metastasis,and/or poor clinical response to the treatment method. In someembodiments, an increased number (such as at least about any one of 1.5,2, 3, 4, 5, 10, or more fold increase) of single CTCs of the individualafter receiving the treatment compared to before receiving the treatmentindicates poor clinical response to the treatment method. In someembodiments, the number of CTC clusters in a blood sample of theindividual is determined. In some embodiments, detection of at leastabout any of 1, 5, 10, 50, 100, or more CTC clusters in a blood sampleof the individual after receiving the treatment indicates an increasedrisk of metastasis, and/or poor clinical response to the treatment. Insome embodiments, an increased number (such as at least about any one of1.5, 2, 3, 4, 5, 10, or more fold increase) of CTC clusters of theindividual after receiving the treatment compared to before receivingthe treatment indicates poor clinical response to the treatment.

III. Tumor-Specific TCRs

Also provided herein are tumor-specific TCRs obtained using any one ofthe methods described in Section II. In some embodiments, thetumor-specific TCR specifically recognizes CEA, RSG-5 or HPV 18-E7.Nucleic acids and vectors encoding the tumor-specific TCRs, engineeredimmune cells expressing the tumor-specific TCRs are also within thescope of this application.

Exemplary Tumor-Specific TCRs

Exemplary TCRs identified using the methods described herein are shownin Table 1 below. The V, J, C segments are named according to IMGTdatabase. Other nomenclature and segment delineation algorithms known inthe art may be used. For example, according to IMGT, a TCR chaincomprises a FR1 from amino acid position 1 to 26, a CDR1 from amino acidposition 27 to 38, a FR2 from amino acid position 39 to 55, a CDR2 fromamino acid position 56 to 65, a FR3 from amino acid position 66 to 104,and a CDR3 from amino acid position 105 to 117 (for rearranged V-J-Genesand V-D-J-genes), and a FR4 from amino acid position 118 to 129. See,for example, Lefranc, M.-P., The Immunologist, 7, 132-136 (1999), andworld wideweb.imgt.org/IMGTScientificChart/Nomenclature/IMGT-FRCDRdefinition.html.CDR1, CDR2 and CDR3 of exemplary TCRs are shown in the “SEQUENCELISTING” section and in Table 5.

TABLE 1 Exemplary tumor-specific TCRs TCR ID Clone # Antigen TCR chain VJ C SEQ ID NO. Epitope CDR3 Full chain Variable reg on amino acidnucleic acid amino acid nucleic acid 1 C E A Alpha TRAV26-1*01 TRAJ48*01Cα 4 5 6 Beta TRBV18*01 TRBJ1-2*01 Cβ1 7 8 9 2 Alpha TRAV16*01 TRAJ27*01Cα 10 11 12 Beta TRBV2*01 TRBJ1-3*01 Cβ1 13 14 15 3 Alpha TRAV23/DV6* 01TRAJ20*01 Cα 16 17 18 Beta TRBV18*01 TRBJ1-4*01 Cβ1 19 20 21 1 R G S 5Alpha TRAV38-2/DV8*01 TRAJ20*01 Cα 82 22 23 24 Beta TRBV19*01 TRBJ2-1*01Cβ2 25 26 27 P09 E06 2 Alpha TRAV26-1*01 TRAJ43*01 Cα 28 29 30 214 215Beta TRBV11-2*01 TRBJ1-1*01 Cβ1 31 32 33 212 213 09D 01 3 AlphaTRAV12-1*01 TRAJ12*01 Cα 82 34 35 36 242 243 Beta TRBV20-1*05 TRBJ2-3*01Cβ2 37 38 39 240 241 09H 05 4 Alpha TRAV12-1*01 TRAJ12*01 Cα 82 40 41 42238 239 Beta TRBV20-1*05 TRBJ2-5*01 Cβ2 43 44 45 236 237 09E 01 5 AlphaTRAV25*01 TRAJ49*01 Cα 83 46 47 48 222 223 Beta TRBV6-5*01 TRBJ1-5*01Cβ1 49 50 51 220 221 09B 03 6 Alpha TRAV16*01 TRAJ23 *01 Cα 82 52 53 54210 211 Beta TRBV30*01^(#) TRBJ2-2*01 Cβ2 55 56 57 208 209 P09 B08 1 H PV 18 E 7 Alpha TRAV12-3 TRAJ38*01 Cα 85 58 59 60 234 235 Beta TRBV19*01TRBJ2-1*01 Cβ2 61 62 63 232 233 2 Alpha TRAV16*01 TRAJ43 *01 Cα 64 65 66Beta TRBV7-9*01 TRBJ2-1*01 Cβ2 67 68 69 10F 04 3 Alpha TRAV12-3*01TRAJ41*01 Cα 85 70 71 72 226 227 Beta TRBV19*01 TRBJ1-4*01 Cβ1 73 74 75224 225 09B 12 4 Alpha TRAV9-2*01 TRAJ53*01 Cα 85 76 77 78 218 219 BetaTRBV9*01 TRBJ2-7*01 Cβ2 79 80 81 216 217 33A 02 5 Alpha TRAV8-3 *02TRAJ54*01 Cα 84 87 88 89 246 247 Beta TRBV20-1*01 TRBJ2-1*01 Cβ2 90 9192 244 245 33D 05 6 Alpha TRAV8-2*01 TRAJ3*01 Cα 86 93 94 95 230 231Beta TRBV7-9*01 TRBJ2-5*01 Cβ2 96 97 98 228 229

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an epitope of a CEA peptidecomprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an epitope of RGS5, such as humanRGS5, for example, amino acids 1-30 or amino acids 16-30 of human RGS5.In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an epitope of a RGS5 peptidecomprising the amino acid sequence of SEQ ID NO: 2. In some embodiments,there is provided an antigen recognizing construct (e.g., a TCR)specifically binds to an epitope of RGS5 comprising the amino acidsequence of SEQ ID NO: 82. In some embodiments, there is provided anantigen recognizing construct (e.g., a TCR) specifically binds to anepitope of RGS5 comprising the amino acid sequence of SEQ ID NO: 83.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an epitope of HPV 18-E7, such ashuman HPV18-E7, for example, amino acids 80-94, 76-105, or 84-102 ofhuman HPV18-E7. In some embodiments, there is provided an antigenrecognizing construct (e.g., a TCR) specifically binds to an epitope ofa HPV18-E7 peptide comprising the amino acid sequence of SEQ ID NO: 3.In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an epitope of HPV18-E7 comprisingthe amino acid sequence of SEQ ID NO: 84. In some embodiments, there isprovided an antigen recognizing construct (e.g., a TCR) specificallybinds to an epitope of HPV18-E7 comprising the amino acid sequence ofSEQ ID NO: 85. In some embodiments, there is provided an antigenrecognizing construct (e.g., a TCR) specifically binds to an epitope ofHPV18-E7 comprising the amino acid sequence of SEQ ID NO: 86.

Also provided are isolated tumor epitopes comprising any one of theamino acid sequences of SEQ ID NOs: 82-86.

In some embodiments, there is provided a tumor-specific antigen bindingconstruct (e.g. tumor-specific TCR) comprising the amino acid sequenceof any one of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37,40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 87, 90, 93 and96. In some embodiments, there is provided a tumor-specific antigenbinding construct (e.g. tumor-specific TCR) comprising: (a) a firstamino acid sequence having at least about 90% sequence identity (e.g.,100% identity) to any one of the amino acid sequences of SEQ ID NOs: 4,10, and 16; and a second amino acid sequence having at least about 90%sequence identity (e.g., 100% identity) to any one of the amino acidsequences of SEQ ID NOs: 7, 13, and 19; (b) a first amino acid sequencehaving at least about 90% sequence identity (e.g., 100% identity) to anyone of the amino acid sequences of SEQ ID NOs: 22, 28, 34, 40, 46, and52; and a second amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to any one of the amino acid sequences ofSEQ ID NOs: 7, 13, 19, 25, 31, 37, 43, 49, and 55; or (c) a first aminoacid sequence having at least about 90% sequence identity (e.g., 100%identity) to any one of the amino acid sequences of SEQ ID NOs: 58, 64,70, 76, 87, and 93; and a second amino acid sequence having at leastabout 90% sequence identity (e.g., 100% identity) to any one of theamino acid sequences of SEQ ID NOs: 61, 67, 73, 79, 90 and 96.

In some embodiments, there is provided a tumor-specific antigen bindingconstruct (e.g. tumor-specific TCR) comprising: (a) a first amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 4, and a second amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 7;(b) a first amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 10, and a second amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 13; (c) a first amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:16, and a second amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 19; (d) a first amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 22, and a second amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:25; (e) a first amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 28, and a second amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 31; (f) a first amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:34, and a second amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 37; (g) a first amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 40, and a second amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:43; (h) a first amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 46, and a second amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 49; (i) a first amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:52, and a second amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 55; (j) a first amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 58, and a second amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:61; (k) a first amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 64, and a second amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 67; (1) a first amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:70, and a second amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 73; (m) a first amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 76, and a second amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:79; (n) a first amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 87, and a second amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 90; or (o) a first amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:93, and a second amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 96. In some embodiments,the antigen-binding construct of any one of (a)-(c) specifically bindsto an epitope of a CEA peptide comprising the amino acid sequence of SEQID NO: 1. In some embodiments, the antigen-binding construct of any oneof (d)-(i) specifically binds to an epitope of a RGS-5 peptidecomprising the amino acid sequence of SEQ ID NO: 2. In some embodiments,the antigen-binding construct of any one of (e)-(g) and (i) specificallybinds to a RGS-5 epitope comprising the amino acid sequence of SEQ IDNO: 82. In some embodiments, the antigen-binding construct of (h)specifically binds to a RGS-5 epitope comprising the amino acid sequenceof SEQ ID NO: 83. In some embodiments, the antigen-binding construct ofany one of (j)-(o) specifically binds an epitope of a HPV18-E7 peptidecomprising the amino acid sequence of SEQ ID NO: 3. In some embodiments,the antigen-binding construct of (n) specifically binds to a HPV18-E7epitope comprising the amino acid sequence of SEQ ID NO: 84. In someembodiments, the antigen-binding construct of any one of (j), (1) and(m) specifically binds to a HPV18-E7 epitope comprising the amino acidsequence of SEQ ID NO: 85. In some embodiments, the antigen-bindingconstruct of (o) specifically binds to a HPV18-E7 epitope comprising theamino acid sequence of SEQ ID NO: 86.

In some embodiments, there is provided a tumor-specific antigen bindingconstruct (e.g. tumor-specific TCR) comprising a CDR3 comprising theamino acid sequence of any one of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22,25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76,79, 87, 90, 93 and 96. In some embodiments, there is provided atumor-specific TCR, comprising: (a) a TCRα chain comprising acomplementary determining region (CDR) 3 having at least about 90%sequence identity (e.g., 100% identity) to any one of the amino acidsequences of SEQ ID NOs: 4, 10, and 16; and a TCRβ chain comprising aCDR3 comprising an amino acid sequence having at least about 90%sequence identity (e.g., 100% identity) to any one of the amino acidsequences of SEQ ID NOs: 7, 13, and 19; (b) a TCRα chain comprising acomplementary determining region (CDR) 3 comprising an amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to any one of the amino acid sequences of SEQ ID NOs: 22, 28,34, 40, 46, and 52; and a TCRβ chain comprising a CDR3 comprising anamino acid sequence having at least about 90% sequence identity (e.g.,100% identity) to any one of the amino acid sequences of SEQ ID NOs: 7,13, 19, 25, 31, 37, 43, 49, and 55; or (c) a TCRα chain comprising acomplementary determining region (CDR) 3 comprising an amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to any one of the amino acid sequences of SEQ ID NOs: 58, 64,70, 76, 87 and 93; and a TCRβ chain comprising a CDR3 comprising anamino acid sequence having at least about 90% sequence identity (e.g.,100% identity) to any one of the amino acid sequences of SEQ ID NOs: 61,67, 73, 79, 90 and 96.

In some embodiments, there is provided a tumor-specific TCR, comprising:(a) a TCRα chain comprising a CDR3 an amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 4,and a TCRβ chain comprising a CDR3 comprising an amino acid sequencehaving at least about 90% sequence identity (e.g., 100% identity) to SEQID NO: 7; (b) a TCRα chain comprising a CDR3 comprising an amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 10, and a TCRβ chain comprising a CDR3comprising an amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 13; (c) a TCRα chaincomprising a CDR3 comprising an amino acid sequence having at leastabout 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 16, anda TCRβ chain comprising a CDR3 comprising an amino acid sequence havingat least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:19; (d) a TCRα chain comprising a CDR3 comprising an amino acid sequencehaving at least about 90% sequence identity (e.g., 100% identity) to SEQID NO: 22, and a TCRβ chain comprising a CDR3 comprising an amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 25; (e) a TCRα chain comprising a CDR3comprising an amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 28, and a TCRβ chaincomprising a CDR3 comprising an amino acid sequence having at leastabout 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 31; (f)a TCRα chain comprising a CDR3 comprising an amino acid sequence havingat least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:34, and a TCRβ chain comprising a CDR3 comprising an amino acid sequencehaving at least about 90% sequence identity (e.g., 100% identity) to SEQID NO: 37; (g) a TCRα chain comprising a CDR3 comprising an amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 40, and a TCRβ chain comprising a CDR3comprising an amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 43; (h) a TCRα chaincomprising a CDR3 comprising an amino acid sequence having at leastabout 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 46, anda TCRβ chain comprising a CDR3 comprising an amino acid sequence havingat least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:49; (i) a TCRα chain comprising a CDR3 comprising an amino acid sequencehaving at least about 90% sequence identity (e.g., 100% identity) to SEQID NO: 52, and a TCRβ chain comprising a CDR3 comprising an amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 55; (j) a TCRα chain comprising a CDR3comprising an amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 58, and a TCRβ chaincomprising a CDR3 comprising an amino acid sequence having at leastabout 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 61;(k) aTCRα chain comprising a CDR3 comprising an amino acid sequence having atleast about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:64, and a TCRβ chain comprising a CDR3 comprising an amino acid sequencehaving at least about 90% sequence identity (e.g., 100% identity) to SEQID NO: 67; (1) a TCRα chain comprising a CDR3 comprising an amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 70, and a TCRβ chain comprising a CDR3comprising an amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 73; (m) a TCRα chaincomprising a CDR3 comprising an amino acid sequence having at leastabout 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 76, anda TCRβ chain comprising a CDR3 comprising an amino acid sequence havingat least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO:79; (n) a TCRα chain comprising a CDR3 comprising an amino acid sequencehaving at least about 90% sequence identity (e.g., 100% identity) to SEQID NO: 87, and a TCRβ chain comprising a CDR3 comprising an amino acidsequence having at least about 90% sequence identity (e.g., 100%identity) to SEQ ID NO: 90; or (o) a TCRα chain comprising a CDR3comprising an amino acid sequence having at least about 90% sequenceidentity (e.g., 100% identity) to SEQ ID NO: 93, and a TCRβ chaincomprising a CDR3 comprising an amino acid sequence having at leastabout 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 96. Insome embodiments, the tumor-specific TCR of any one of (a)-(c)specifically binds to an epitope of a CEA peptide comprising the aminoacid sequence of SEQ ID NO: 1. In some embodiments, the tumor-specificTCR of any one of (d)-(i) specifically binds to an epitope of a RGS-5peptide comprising the amino acid sequence of SEQ ID NO: 2. In someembodiments, the tumor-specific TCR of any one of (e)-(g) and (i)specifically binds to a RGS-5 epitope comprising the amino acid sequenceof SEQ ID NO: 82. In some embodiments, the tumor-specific TCR of (h)specifically binds to a RGS-5 epitope comprising the amino acid sequenceof SEQ ID NO: 83. In some embodiments, the tumor-specific TCR of any oneof (j)-(o) specifically binds an epitope of a HPV18-E7 peptidecomprising the amino acid sequence of SEQ ID NO: 3. In some embodiments,the tumor-specific TCR of (n) specifically binds to a HPV18-E7 epitopecomprising the amino acid sequence of SEQ ID NO: 84. In someembodiments, the tumor-specific TCR of any one of (j), (l) and (m)specifically binds to a HPV18-E7 epitope comprising the amino acidsequence of SEQ ID NO: 85. In some embodiments, the tumor-specific TCRof (o) specifically binds to a HPV18-E7 epitope comprising the aminoacid sequence of SEQ ID NO: 86.

Any of the tumor-specific TCRs described herein may comprise a V elementand/or a J element. Any suitable V and J elements may be applicable,see, for example, the IMGT database. Table 1 shows exemplarycombinations of the V element and J elements. In some embodiments, thetumor-specific TCR comprises: a TCRα chain comprising a TRAV element ofany one of SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71,77, 88, and 94, or a variant thereof; and a TCRβ chain comprising a TRBVelement of any one of the amino acid sequences of SEQ ID NOs: 8, 14, 20,26, 32, 38, 44, 56, 62, 68, 74, 80, 91 and 97 or a variant thereof. Insome embodiments, the tumor-specific TCR comprises: a TCRα chaincomprising a TRAJ element of any one of SEQ ID NOs: 5, 11, 17, 23, 29,35, 41, 47, 53, 59, 65, 71, 77, 88, and 94, or a variant thereof; and aTCRβ chain comprising a TRBJ element of any one of the amino acidsequences of SEQ ID NOs: 8, 14, 20, 26, 32, 38, 44, 56, 62, 68, 74, 80,91 and 97, or a variant thereof.

In some embodiments, there is provided a tumor-specific TCR, comprising:(a) a TCRα chain comprising CDRs of any one of the amino acid sequencesof SEQ ID NOs: 5, 11 and 17, and a TCRβ chain comprising CDRs of any oneof the amino acid sequences of SEQ ID NOs: 8, 14 and 20; (b) a TCRαchain comprising CDRs of any one of the amino acid sequences of SEQ IDNOs: 23, 29, 35, 41, 47 and 53, and a TCRβ chain comprising CDRs of anyone of the amino acid sequences of SEQ ID NOs: 26, 32, 38, 44, 50 and56; or (c) a TCRα chain comprising CDRs of any one of the amino acidsequences of SEQ ID NOs: 59, 65, 71, 77, 88 and 94, and a TCRβ chaincomprising CDRs of any one of the amino acid sequences of SEQ ID NOs:62, 68, 74, 80, 91 and 97.

The tumor-specific TCRs described herein also comprise TCR constantdomains. In some embodiments, the tumor-specific TCR comprises: a TCRαchain comprising a TCRα constant domain (TRAC) of any one of SEQ ID NOs:5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 88 and 94, or avariant thereof; and a TCRβ chain comprising a TCRβ constant domain(TRBC) of any one of the amino acid sequences of SEQ ID NOs: 8, 14, 20,26, 32, 38, 44, 56, 62, 68, 74, 80, 91 and 97, or a variant thereof. Insome embodiments, the tumor-specific TCR comprises a human TRAC and ahuman TRBC, such as human Cα and human Cβ1 or human Cα and human Cβ2. Insome embodiments, the tumor-specific TCR comprises: a TCRα chaincomprising a TRAC comprising the amino acid sequence of SEQ ID NO: 198,or a variant thereof; and a TCRβ chain comprising a TRBC comprising theamino acid sequence of SEQ ID NO: 202 or 204. In some embodiments, thetumor-specific TCR comprises a murine TRAC and a murine TRBC, such asmurine Cα and murine Cβ1. In some embodiment, the murine TRAC comprisesa modification at position 117 (e.g., S117L) and/or 110 (e.g., G110V).In some embodiments, the tumor-specific TCR comprises: a TCRα chaincomprising a TRAC comprising the amino acid sequence of SEQ ID NO: 200,or a variant thereof; and a TCRβ chain comprising a TRBC comprising theamino acid sequence of SEQ ID NO: 206.

In some embodiments, the tumor-specific TCR is a human TCR. In someembodiments, the tumor-specific TCR is a chimeric TCR, such as amurinized TCR, e.g., a TCR comprising murine constant regions of TCRαand β chains. In some embodiments, the tumor-specific TCR compriseshuman TCR variable regions and TCR constant regions from a non-humanspecies, such as mouse.

In some embodiments, there is provided a tumor-specific TCR, comprising:(a) a TCRα chain comprising an amino acid sequence having at least about80% identity (e.g., at least about any one of 85%, 90%, 95%, 98% orhigher identity, or having 100% identity) to any one of the amino acidsequences of SEQ ID NOs: 5, 11 and 17, and a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to any one of the amino acid sequences of SEQ ID NOs: 8, 14and 20; (b) a TCRα chain comprising an amino acid sequence having atleast about 80% identity (e.g., at least about any one of 85%, 90%, 95%,98% or higher identity, or having 100% identity) to any one of the aminoacid sequences of SEQ ID NOs: 23, 29, 35, 41, 47 and 53, and a TCRβchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to any one of the amino acidsequences of SEQ ID NOs: 26, 32, 38, 44, 50 and 56; or (c) a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to any one of the amino acid sequences of SEQID NOs: 59, 65, 71, 77, 88 and 94, and a TCRβ chain comprising an aminoacid sequence having at least about 80% identity (e.g., at least aboutany one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to any one of the amino acid sequences of SEQ ID NOs: 62, 68,74, 80 and 91 and 97.

In some embodiments, there is provided a tumor-specific TCR, comprising:(a) a TCRα chain comprising an amino acid sequence having at least about80% identity (e.g., at least about any one of 85%, 90%, 95%, 98% orhigher identity, or having 100% identity) to SEQ ID NO: 5; a TCRβ chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 8; (b) a TCRα chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 11; a TCRβ chain comprising an amino acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 14; (c) a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:17; a TCRβ chain comprising an amino acid sequence having at least about80% identity (e.g., at least about any one of 85%, 90%, 95%, 98% orhigher identity, or having 100% identity) to SEQ ID NO: 20; (d) a TCRαchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 23; a TCRβ chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 26; (e) a TCRα chain comprisingan amino acid sequence having at least about 80% identity (e.g., atleast about any one of 85%, 90%, 95%, 98% or higher identity, or having100% identity) to SEQ ID NO: 29; a TCRβ chain comprising an amino acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 32; (f) a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:35; a TCRβ chain comprising an amino acid sequence having at least about80% identity (e.g., at least about any one of 85%, 90%, 95%, 98% orhigher identity, or having 100% identity) to SEQ ID NO: 38; (g) a TCRαchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 41; a TCRβ chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 44; (h) a TCRα chain comprisingan amino acid sequence having at least about 80% identity (e.g., atleast about any one of 85%, 90%, 95%, 98% or higher identity, or having100% identity) to SEQ ID NO: 47; a TCRβ chain comprising an amino acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 50; (i) a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:53; a TCRβ chain comprising an amino acid sequence having at least about80% identity (e.g., at least about any one of 85%, 90%, 95%, 98% orhigher identity, or having 100% identity) to SEQ ID NO: 56; (j) a TCRαchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 59; a TCRβ chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 62; (k) a TCRα chain comprisingan amino acid sequence having at least about 80% identity (e.g., atleast about any one of 85%, 90%, 95%, 98% or higher identity, or having100% identity) to SEQ ID NO: 65; a TCRβ chain comprising an amino acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 68; (l) a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:71; a TCRβ chain comprising an amino acid sequence having at least about80% identity (e.g., at least about any one of 85%, 90%, 95%, 98% orhigher identity, or having 100% identity) to SEQ ID NO: 74; (m) a TCRαchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 77; a TCRβ chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 80; (n) a TCRα chain comprisingan amino acid sequence having at least about 80% identity (e.g., atleast about any one of 85%, 90%, 95%, 98% or higher identity, or having100% identity) to SEQ ID NO: 88; a TCRβ chain comprising an amino acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 91; or (o) a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:94; a TCRβ chain comprising an amino acid sequence having at least about80% identity (e.g., at least about any one of 85%, 90%, 95%, 98% orhigher identity, or having 100% identity) to SEQ ID NO: 97. In someembodiments, the tumor-specific TCR of any one of (a)-(c) specificallybinds to an epitope of a CEA peptide comprising the amino acid sequenceof SEQ ID NO: 1. In some embodiments, the tumor-specific TCR of any oneof (d)-(i) specifically binds to an epitope of a RGS-5 peptidecomprising the amino acid sequence of SEQ ID NO: 2. In some embodiments,the tumor-specific TCR of any one of (e)-(g) and (i) specifically bindsto a RGS-5 epitope comprising the amino acid sequence of SEQ ID NO: 82.In some embodiments, the tumor-specific TCR of (h) specifically binds toa RGS-5 epitope comprising the amino acid sequence of SEQ ID NO: 83. Insome embodiments, the tumor-specific TCR of any one of (j)-(o)specifically binds an epitope of a HPV18-E7 peptide comprising the aminoacid sequence of SEQ ID NO: 3. In some embodiments, the tumor-specificTCR of (n) specifically binds to a HPV18-E7 epitope comprising the aminoacid sequence of SEQ ID NO: 84. In some embodiments, the tumor-specificTCR of any one of (j), (l) and (m) specifically binds to a HPV18-E7epitope comprising the amino acid sequence of SEQ ID NO: 85. In someembodiments, the tumor-specific TCR of (o) specifically binds to aHPV18-E7 epitope comprising the amino acid sequence of SEQ ID NO: 86.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/RGS5 epitope complex, whereinthe RGS5 epitope comprises the amino acid sequence of SEQ ID NO: 82, andwherein the MHC is HLA-DPA1*02:02/DPB1*05:01. In some embodiments, theantigen recognizing construct comprises a TCRα chain comprising a CDR3comprising SEQ ID NO: 28, and a TCRβ chain comprising a CDR3 comprisingSEQ ID NO: 31. In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising a CDR1 comprising the amino acidsequence of SEQ ID NO: 254, a CDR2 comprising the amino acid sequence ofSEQ ID NO: 255, and a CDR3 comprising the amino acid sequence of SEQ IDNO: 28; and a TCRβ chain comprising a CDR1 comprising the amino acidsequence of SEQ ID NO: 256, a CDR2 comprising the amino acid sequence ofSEQ ID NO: 257, and a CDR3 comprising the amino acid sequence of SEQ IDNO: 31. In some embodiments, the antigen recognizing construct comprisesa TCRα chain comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 214; a TCRβchain comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 212. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 214; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 212. In some embodiments, the antigenrecognizing construct is a human TCR. In some embodiments, the antigenrecognizing construct is a chimeric TCR, such as murinized TCR, e.g., aTCR comprising murine constant regions of TCRα and β chains. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 29; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 32. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:103; a TCRβ chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 105. In someembodiments, the antigen recognizing construct comprises the amino acidsequence of SEQ ID NO: 147 or 149.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/RGS5 epitope complex, whereinthe RGS5 epitope comprises the amino acid sequence of SEQ ID NO: 82, andwherein the MHC is HLA-DRA/DRB1*09:01 or HLA-DRA/DRB4*01:03. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising a CDR3 comprising SEQ ID NO: 34, and a TCRβ chain comprisinga CDR3 comprising SEQ ID NO: 37. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 258, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 259, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 34; and a TCRβ chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 260, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 261, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 37. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising CDR1, CDR2 andCDR3 of SEQ ID NO: 242; a TCRβ chain comprising CDR1, CDR2 and CDR3 ofSEQ ID NO: 240.In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 242; a TCRβchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 240. In someembodiments, the antigen recognizing construct is a human TCR. In someembodiments, the antigen recognizing construct is a chimeric TCR, suchas murinized TCR, e.g., a TCR comprising murine constant regions of TCRαand β chains. In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 35; a TCRβchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 38. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 107; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 109. In some embodiments, the antigenrecognizing construct comprises the amino acid sequence of any one ofSEQ ID NOs: 151, 153, 155 and 157.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/RGS5 epitope complex, whereinthe RGS5 epitope comprises the amino acid sequence of SEQ ID NO: 82, andwherein the MHC is HLA-DRA/DRB1*09:01 or HLA-DRA/DRB4*01:03. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising a CDR3 comprising SEQ ID NO: 40, and a TCRβ chain comprisinga CDR3 comprising SEQ ID NO: 43. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 262, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 263, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 40; and a TCRβ chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 264, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 265, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 43. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising CDR1, CDR2 andCDR3 of SEQ ID NO: 238; a TCRβ chain comprising CDR1, CDR2 and CDR3 ofSEQ ID NO: 236. In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 238; a TCRβchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 236. In someembodiments, the antigen recognizing construct is a human TCR. In someembodiments, the antigen recognizing construct is a chimeric TCR, suchas murinized TCR, e.g., a TCR comprising murine constant regions of TCRαand β chains. In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 41; a TCRβchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 44. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 111; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 113. In some embodiments, the antigenrecognizing construct comprises the amino acid sequence of any one ofSEQ ID NOs: 159, 161, 163 and 165.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/RGS5 epitope complex, whereinthe RGS5 epitope comprises the amino acid sequence of SEQ ID NO: 83, andwherein the MHC is HLA-DRA/DRB1*09:01 or HLA-DRA/DRB4*01:03. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising a CDR3 comprising SEQ ID NO: 46, and a TCRβ chain comprisinga CDR3 comprising SEQ ID NO: 49. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 266, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 267, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 46; and a TCRβ chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 268, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 269, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 49. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising CDR1, CDR2 andCDR3 of SEQ ID NO: 222; a TCRβ chain comprising CDR1, CDR2 and CDR3 ofSEQ ID NO: 220. In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 222; a TCRβchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 220. In someembodiments, the antigen recognizing construct is a human TCR. In someembodiments, the antigen recognizing construct is a murinized TCR, e.g.,a TCR comprising murine constant regions of TCRα and β chains. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 47; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 50. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:115; a TCRβ chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 117. In someembodiments, the antigen recognizing construct comprises the amino acidsequence of any one of SEQ ID NOs: 167 and 169.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/RGS5 epitope complex, whereinthe RGS5 epitope comprises the amino acid sequence of SEQ ID NO: 82, andwherein the MHC is HLA-DPA1*02:02/DPB1*05:01. In some embodiments, theantigen recognizing construct comprises a TCRα chain comprising a CDR3comprising SEQ ID NO: 52, and a TCRβ chain comprising a CDR3 comprisingSEQ ID NO: 55. In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising a CDR1 comprising the amino acidsequence of SEQ ID NO: 270, a CDR2 comprising the amino acid sequence ofSEQ ID NO: 271, and a CDR3 comprising the amino acid sequence of SEQ IDNO: 52; and a TCRβ chain comprising a CDR1 comprising the amino acidsequence of SEQ ID NO: 272, a CDR2 comprising the amino acid sequence ofSEQ ID NO: 273, and a CDR3 comprising the amino acid sequence of SEQ IDNO: 55. In some embodiments, the antigen recognizing construct comprisesa TCRα chain comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 210; a TCRβchain comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 208. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 210; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 208. In some embodiments, the antigenrecognizing construct is a human TCR. In some embodiments, the antigenrecognizing construct is a chimeric TCR, such as murinized TCR, e.g., aTCR comprising murine constant regions of TCRα and β chains. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 53; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 56. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:119; a TCRβ chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 121. In someembodiments, the antigen recognizing construct comprises the amino acidsequence of any one of SEQ ID NOs: 143 and 145.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/HPV18-E7 epitope complex,wherein the HPV18-E7 epitope comprises the amino acid sequence of SEQ IDNO: 85, and wherein the MHC is HLA-DRA/DRB1*09:01. In some embodiments,the antigen recognizing construct comprises a TCRα chain comprising aCDR3 comprising SEQ ID NO: 58, and a TCRβ chain comprising a CDR3comprising SEQ ID NO: 61. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising a CDR1 comprising the aminoacid sequence of SEQ ID NO: 274, a CDR2 comprising the amino acidsequence of SEQ ID NO: 275, and a CDR3 comprising the amino acidsequence of SEQ ID NO: 58; and a TCRβ chain comprising a CDR1 comprisingthe amino acid sequence of SEQ ID NO: 276, a CDR2 comprising the aminoacid sequence of SEQ ID NO: 277, and a CDR3 comprising the amino acidsequence of SEQ ID NO: 61. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising CDR1, CDR2 and CDR3 of SEQID NO: 234; a TCRβ chain comprising CDR1, CDR2 and CDR3 of SEQ ID NO:232. In some embodiments, the antigen recognizing construct comprises aTCRα chain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 234; a TCRβ chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 232. In some embodiments, theantigen recognizing construct is a human TCR. In some embodiments, theantigen recognizing construct is a chimeric TCR, such as a murinizedTCR, e.g., a TCR comprising murine constant regions of TCRα and βchains. In some embodiments, the antigen recognizing construct comprisesa TCRα chain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 59; a TCRβ chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 62. In some embodiments, theantigen recognizing construct comprises a TCRα chain comprising an aminoacid sequence having at least about 80% identity (e.g., at least aboutany one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 123; a TCRβ chain comprising an amino acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 125. In some embodiments, the antigen recognizingconstruct comprises the amino acid sequence of any one of SEQ ID NOs:179 and 181.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/HPV18-E7 epitope complex,wherein the HPV18-E7 epitope comprises the amino acid sequence of SEQ IDNO: 85, and wherein the MHC is HLA-DRA/DRB1*09:01 or HLA-DRA/DRB4*01:03.In some embodiments, the antigen recognizing construct comprises a TCRαchain comprising a CDR3 comprising SEQ ID NO: 70, and a TCRβ chaincomprising a CDR3 comprising SEQ ID NO: 73. In some embodiments, theantigen recognizing construct comprises a TCRα chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 278, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 279, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 70; and a TCRβ chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 280, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 281, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 73. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising CDR1, CDR2 andCDR3 of SEQ ID NO:226; a TCRβ chain comprising CDR1, CDR2 and CDR3 ofSEQ ID NO: 224. In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 226; a TCRβchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 224. In someembodiments, the antigen recognizing construct is a human TCR. In someembodiments, the antigen recognizing construct is a chimeric TCR, suchas a murinized TCR, e.g., a TCR comprising murine constant regions ofTCRα and β chains. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:71; a TCRβ chain comprising an amino acid sequence having at least about80% identity (e.g., at least about any one of 85%, 90%, 95%, 98% orhigher identity, or having 100% identity) to SEQ ID NO: 74. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 127; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 129. In some embodiments, the antigenrecognizing construct comprises the amino acid sequence of any one ofSEQ ID NOs: 183 and 185.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/HPV18-E7 epitope complex,wherein the HPV18-E7 epitope comprises the amino acid sequence of SEQ IDNO: 85, and wherein the MHC is HLA-II. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising a CDR3comprising SEQ ID NO: 76, and a TCRβ chain comprising a CDR3 comprisingSEQ ID NO: 79. In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising a CDR1 comprising the amino acidsequence of SEQ ID NO: 282, a CDR2 comprising the amino acid sequence ofSEQ ID NO: 283, and a CDR3 comprising the amino acid sequence of SEQ IDNO: 76; and a TCRβ chain comprising a CDR1 comprising the amino acidsequence of SEQ ID NO: 284, a CDR2 comprising the amino acid sequence ofSEQ ID NO: 285, and a CDR3 comprising the amino acid sequence of SEQ IDNO: 79. In some embodiments, the antigen recognizing construct comprisesa TCRα chain comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 218; a TCRβchain comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 216. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 218; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 216. In some embodiments, the antigenrecognizing construct is a human TCR. In some embodiments, the antigenrecognizing construct is a chimeric TCR, such as a murinized TCR, e.g.,a TCR comprising murine constant regions of TCRα and β chains. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 77; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 80. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:131; a TCRβ chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 133. In someembodiments, the antigen recognizing construct comprises the amino acidsequence of any one of SEQ ID NOs: 187 and 189.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/HPV18-E7 epitope complex,wherein the HPV18-E7 epitope comprises the amino acid sequence of SEQ IDNO: 84, and wherein the MHC is HLA-DRA/DRB1*09:01. In some embodiments,the antigen recognizing construct comprises a TCRα chain comprising aCDR3 comprising SEQ ID NO: 87, and a TCRβ chain comprising a CDR3comprising SEQ ID NO: 90. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising a CDR1 comprising the aminoacid sequence of SEQ ID NO: 286, a CDR2 comprising the amino acidsequence of SEQ ID NO: 287, and a CDR3 comprising the amino acidsequence of SEQ ID NO: 87; and a TCRβ chain comprising a CDR1 comprisingthe amino acid sequence of SEQ ID NO: 288, a CDR2 comprising the aminoacid sequence of SEQ ID NO: 289, and a CDR3 comprising the amino acidsequence of SEQ ID NO: 90. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising CDR1, CDR2 and CDR3 of SEQID NO: 246; a TCRβ chain comprising CDR1, CDR2 and CDR3 of SEQ ID NO:244. In some embodiments, the antigen recognizing construct comprises aTCRα chain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 246; a TCRβ chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 244. In some embodiments, theantigen recognizing construct is a human TCR. In some embodiments, theantigen recognizing construct is a chimeric TCR, such as a murinizedTCR, e.g., a TCR comprising murine constant regions of TCRα and βchains. In some embodiments, the antigen recognizing construct comprisesa TCRα chain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 88; a TCRβ chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 91. In some embodiments, theantigen recognizing construct comprises a TCRα chain comprising an aminoacid sequence having at least about 80% identity (e.g., at least aboutany one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 135; a TCRβ chain comprising an amino acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 137. In some embodiments, the antigen recognizingconstruct comprises the amino acid sequence of any one of SEQ ID NOs:171, 173, 175 and 177.

In some embodiments, there is provided an antigen recognizing construct(e.g., a TCR) specifically binds to an MHC/HPV18-E7 epitope complex,wherein the HPV18-E7 epitope comprises the amino acid sequence of SEQ IDNO: 86, and wherein the MHC is HLA-DPA1*02:02/DPB1*05:01,HLA-DPA1*01:03/DPB1*02:01, or HLA-DPA1*01:03/DPB1*05:01. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising a CDR3 comprising SEQ ID NO: 93, and a TCRβ chain comprisinga CDR3 comprising SEQ ID NO: 96. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 290, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 291, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 93; and a TCRβ chain comprising a CDR1comprising the amino acid sequence of SEQ ID NO: 292, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 293, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 96. In some embodiments, the antigenrecognizing construct comprises a TCRα chain comprising CDR1, CDR2 andCDR3 of SEQ ID NO: 230; a TCRβ chain comprising CDR1, CDR2 and CDR3 ofSEQ ID NO: 228. In some embodiments, the antigen recognizing constructcomprises a TCRα chain comprising an amino acid sequence having at leastabout 80% identity (e.g., at least about any one of 85%, 90%, 95%, 98%or higher identity, or having 100% identity) to SEQ ID NO: 230; a TCRβchain comprising an amino acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 228. In someembodiments, the antigen recognizing construct is a human TCR. In someembodiments, the antigen recognizing construct is a chimeric TCR, suchas a murinized TCR, e.g., a TCR comprising murine constant regions ofTCRα and β chains. In some embodiments, the antigen recognizingconstruct comprises a TCRα chain comprising an amino acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:94; a TCRβ chain comprising an amino acid sequence having at least about80% identity (e.g., at least about any one of 85%, 90%, 95%, 98% orhigher identity, or having 100% identity) to SEQ ID NO: 97. In someembodiments, the antigen recognizing construct comprises a TCRα chaincomprising an amino acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 139; a TCRβ chain comprising anamino acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 141. In some embodiments, the antigenrecognizing construct comprises the amino acid sequence of any one ofSEQ ID NOs: 191 and 193.

Naturally occurring TCRs are heterodimeric receptors composed of aβ orγδ chains that pair on the surface of a T cell. Each α, β, γ, and δchain is composed of two Ig-like domains: a variable domain (V) thatconfers antigen recognition through the complementarity determiningregions (CDR), followed by a constant domain (C) that is anchored tocell membrane by a connecting peptide and a transmembrane (TM) region.The TM region associates with the invariant subunits of the CD3signaling apparatus. Each of the V domains has three CDRs. These CDRsinteract with a complex between an antigenic peptide bound to a proteinencoded by the major histocompatibility complex (pMHC) (Davis andBjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu RevImmunol, 16, 523-544; Murphy (2012), xix, 868 p.).

“Homology” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are“homologous” at that position. The “percent of homology” or “percentsequence identity” between two sequences is a function of the number ofmatching or homologous positions shared by the two sequences divided bythe number of positions compared times 100, considering any conservativesubstitutions as part of the sequence identity. For example, if 6 of 10of the positions in two sequences are matched or homologous then the twosequences are 60% homologous. By way of example, the DNA sequencesATTGCC and TATGGC share 50% homology. Generally, a comparison is madewhen two sequences are aligned to give maximum homology. Alignment forpurposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are generated using thesequence comparison computer program MUSCLE (Edgar, R.C., Nucleic AcidsResearch 32(5): 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics5(1):113, 2004).

Also provided are epitopes, and MHC/epitope complexes that any one ofthe TCRs described herein recognizes. Further provided are TCRs thatcompetitively bind to the same MHC/epitope complex as any one of theTCRs described herein.

HLA Restriction

The tumor-specific TCRs described herein are MHC class I or MHC class IIrestricted. In some embodiments, the tumor-specific TCR is restricted toa HLA haplotype. In some embodiments, the tumor-specific TCR has a HLAhaplotype restriction that is predominant in Asians. FIGS. 25A-25B showHLA restrictions of exemplary TCRs.

MHC class I proteins are one of two primary classes of majorhistocompatibility complex (MHC) molecules (the other being MHC classII) and are found on nearly every nucleated cell of the body. Theirfunction is to display fragments of proteins from within the cell to Tcells; healthy cells will be ignored, while cells containing foreign ormutated proteins will be attacked by the immune system. Because MHCclass I proteins present peptides derived from cytosolic proteins, thepathway of MHC class I presentation is often called the cytosolic orendogenous pathway. Class I MHC molecules bind peptides generated mainlyfrom degradation of cytosolic proteins by the proteasome. The MHCI:peptide complex is then inserted into the plasma membrane of the cell.The peptide is bound to the extracellular part of the class I MHCmolecule. Thus, the function of the class I MHC is to displayintracellular proteins to cytotoxic T cells (CTLs). However, class I MHCcan also present peptides generated from exogenous proteins, in aprocess known as cross-presentation.

MHC class I proteins consist of two polypeptide chains, α andβ2-microglobulin (β2M). The two chains are linked noncovalently viainteraction of β2M and the α3 domain. Only the α chain is polymorphicand encoded by a HLA gene, while the β2M subunit is not polymorphic andencoded by the β-2 microglobulin gene. The α3 domain is plasmamembrane-spanning and interacts with the CD8 co-receptor of T-cells. Theα3-CD8 interaction holds the MHC I molecule in place while the T cellreceptor (TCR) on the surface of the cytotoxic T cell binds its α1-α2heterodimer ligand, and checks the coupled peptide for antigenicity. Theα1 and α2 domains fold to make up a groove for peptides to bind. MHCclass I proteins bind peptides that are 8-10 amino acid in length.

MHC class II molecules are a family of molecules normally found only onantigen-presenting cells such as dendritic cells, mononuclearphagocytes, some endothelial cells, thymic epithelial cells, and Bcells. The antigens presented by class II peptides are derived fromextracellular proteins (not cytosolic as in class I); hence, the MHCclass II-dependent pathway of antigen presentation is called theendocytic or exogenous pathway. Loading of an MHC class II moleculeoccurs by phagocytosis; extracellular proteins are endocytosed, digestedin lysosomes, and the resulting epitopic peptide fragments are loadedonto MHC class II molecules prior to their migration to the cellsurface.

Like MHC class I molecules, class II molecules are also heterodimers,but in this case consist of two homogenous peptides, an α and β chain.The subdesignation α1, α2, etc. refers to separate domains within theHLA gene; each domain is usually encoded by a different exon within thegene, and some genes have further domains that encode leader sequences,transmembrane sequences, etc. Because the antigen-binding groove of MHCclass II molecules is open at both ends while the corresponding grooveon class I molecules is closed at each end, the antigens presented byMHC class II molecules are longer, generally between 15 and 24 aminoacid residues long.

The human leukocyte antigen (HLA) genes are the human versions of theMHC genes. The three major MHC class I proteins in humans are HLA-A,HLA-B, and HLA-C, while the 3 minor ones are HLA-E, HLA-F, and HLA-G.The three major MHC class II proteins involved in antigen presentationin humans are HLA-DP, HLDA-DQ, and HLA-DR, while the other MHC class IIproteins, HLA-DM and HLA-DO, are involved in the internal processing andloading of antigens. HLA-A is ranked among the genes in humans with thefastest-evolving coding sequence. As of December 2013, there were 2432known HLA-A alleles coding for 1740 active proteins and 117 nullproteins. The HLA-A gene is located on the short arm of chromosome 6 andencodes the larger, α-chain, constituent of HLA-A. Variation of HLA-Aα-chain is key to HLA function. This variation promotes geneticdiversity in the population. Since each HLA has a different affinity forpeptides of certain structures, greater variety of HLAs means greatervariety of antigens to be ‘presented’ on the cell surface, enhancing thelikelihood that a subset of the population will be resistant to anygiven foreign invader. This decreases the likelihood that a singlepathogen has the capability to wipe out the entire human population.Each individual can express up to two types of HLA-A, one from each oftheir parents. Some individuals will inherit the same HLA-A from bothparents, decreasing their individual HLA diversity; however, themajority of individuals will receive two different copies of HLA-A. Thissame pattern follows for all HLA groups. In other words, a person canonly express either one or two of the 2432 known HLA-A alleles.

All alleles receive at least a four digit classification, e.g.,HLA-A*02:12. The A signifies which HLA gene the allele belongs to. Thereare many HLA-A alleles, so that classification by serotype simplifiescategorization. The next pair of digits indicates this assignment. Forexample, HLA-A*02:02, HLA-A*02:04, and HLA-A*02:324 are all members ofthe A2 serotype (designated by the *02 prefix). This group is theprimary factor responsible for HLA compatibility. All numbers after thiscannot be determined by serotyping and are designated through genesequencing. The second set of digits indicates what HLA protein isproduced. These are assigned in order of discovery and as of December2013 there are 456 different HLA-A02 proteins known (assigned namesHLA-A*02:01 to HLA-A*02:456). The shortest possible HLA name includesboth of these details. Each extension beyond that signifies a nucleotidechange that may or may not change the protein.

In some embodiments, the tumor-specific TCR specifically binds to acomplex comprising the target tumor antigen peptide and an MHC class Iprotein, wherein the MHC class I protein is HLA-A, HLA-B, HLA-C, HLA-E,HLA-F, or HLA-G. In some embodiments, the MHC class I protein is HLA-A,HLA-B, or HLA-C. In some embodiments, the MHC class I protein is HLA-A.In some embodiments, the MHC class I protein is HLA-B. In someembodiments, the MHC class I protein is HLA-C. In some embodiments, theMHC class I protein is HLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10,HLA-A11, HLA-A19, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29,HLA-A30, HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66,HLA-A68, HLA-A69, HLA-A74, or HLA-A80. In some embodiments, the MHCclass I protein is HLA-A02. In some embodiments, the MHC class I proteinis any one of HLA-A*02:01-555, such as HLA-A*02:01, HLA-A*02:02,HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07,HLA-A*02:08, HLA-A*02:09, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12,HLA-A*02:13, HLA-A*02:14, HLA-A*02:15, HLA-A*02:16, HLA-A*02:17,HLA-A*02:18, HLA-A*02:19, HLA-A*02:20, HLA-A*02:21, HLA-A*02:22, orHLA-A*02:24. In some embodiments, the MHC class I protein isHLA-A*02:01. HLA-A*02:01 is expressed in 39-46% of all Caucasians.Tumor-specific TCRs having HLA-A*02:01 restriction may be especiallysuitable for treating Caucasian patients. In some embodiments, the MHCclass I protein is HLA-A* 1101. HLA-A* 1101 is expressed in about 25-30%of all Chinese. Tumor-specific TCRs having HLA-A*1101 restriction may beespecially suitable for treating Chinese patients.

In some embodiments, the tumor-specific TCR specifically binds to acomplex comprising the target tumor antigen peptide and an MHC class IIprotein, wherein the MHC class II protein is HLA-DP, HLA-DQ, or HLA-DR.In some embodiments, the MHC class II protein is HLA-DP. In someembodiments, the MHC class II protein is HLA-DQ. In some embodiments,the MHC class II protein is HLA-DR. In some embodiments, the MHC classII protein is HLA-DPB1*0401. HLA-DPB1*0401 is expressed in 35-40% of allCaucasians. Tumor-specific TCRs having HLA-DPB1*0401 restriction may beespecially suitable for treating Caucasian patients.

Variants

In some embodiments, amino acid sequence variants of the tumor-specificantigen binding constructs (e.g., tumor-specific TCRs) provided hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of thetumor-specific antigen binding constructs (e.g., tumor-specific TCRs).Amino acid sequence variants of a tumor-specific TCR may be prepared byintroducing appropriate modifications into the nucleotide sequence(s)encoding the TCRα or TCRβ chain of the tumor-specific TCR, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of residues within the aminoacid sequences of the tumor-specific TCR. Any combination of deletion,insertion, and substitution can be made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics, e.g., antigen-binding.

In some embodiments, tumor-specific antigen binding construct (e.g.,tumor-specific TCR) variants having one or more amino acid substitutionsare provided. Sites of interest for substitutional mutagenesis includethe CDRs and FRs. Amino acid substitutions may be introduced into atumor-specific TCR of interest and the products screened for a desiredactivity, e.g., retained/improved antigen binding or decreasedimmunogenicity.

Conservative substitutions are shown in Table 2 below.

TABLE 2 CONSERVATIVE SUBSTITITIONS Original Residue ExemplarySubstitutions Preferred Substitutions Ala (A) Val; Leu; Ile Val Arg (R)Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; AsnGlu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly(G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala;Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val;Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser SerTrp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu;Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped into different classes according to commonside-chain properties:

-   a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;-   b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;-   c. acidic: Asp, Glu;-   d. basic: His, Lys, Arg;-   e. residues that influence chain orientation: Gly, Pro;-   f. aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

An exemplary substitutional variant is an affinity maturedtumor-specific TCR, which may be conveniently generated, e.g., usingphage display-based affinity maturation techniques. Briefly, one or moreCDR residues are mutated and the variant tumor-specific TCR moietiesdisplayed on phage and screened for a particular biological activity(e.g., binding affinity). Alterations (e.g., substitutions) may be madein CDRs, e.g., to improve tumor-specific TCR affinity.

In some embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any tumor-specific TCR variants with the desiredaffinity. Another method to introduce diversity involves CDR-directedapproaches, in which several CDR residues (e.g., 4-6 residues at a time)are randomized. CDR residues involved in antigen binding may bespecifically identified, e.g., using alanine scanning mutagenesis ormodeling.

In some embodiments, substitutions, insertions, or deletions may occurwithin one or more CDRs so long as such alterations do not substantiallyreduce the ability of the tumor-specific TCR to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in CDRs. In some embodiments of the variant TCRα or TCRβsequences provided above, each CDR either is unaltered, or contains nomore than one, two or three amino acid substitutions.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. In some embodiments, the tumor-specificTCR construct comprises a leader sequence at the N-terminus of the TCRαchain or the TCRβ chain. In some embodiments, the leader sequencecomprises the amino acid sequence of SEQ ID NO: 101.

In some embodiments, there is provided a tumor specific TCR constructcomprising a polypeptide comprising a TCRα chain polypeptide, aself-cleavable linker, and a TCRβ chain polypeptide. In someembodiments, the self-cleavable linker is a P2A peptide, a T2A peptide,a E2A peptide, or a F2A peptide. In some embodiments, the self-cleavablelinker comprises any one of the amino acid sequences of SEQ ID NOs: 99,195, 196 and 197.

Antigen binding fragments and derivatives of the tumor-specific TCRsdescribed herein are also contemplated.

Nucleic Acids

In some embodiments, according to any of the tumor-specific TCRsdescribed herein, there is provided a nucleic acid (or a set of nucleicacids) encoding the tumor-specific TCR. The present invention alsoprovides vectors in which one or more nucleic acids of the presentinvention is incorporated.

In some embodiments, there is provided an isolated nucleic acid encodingthe tumor-specific TCR or components or derivatives thereof (such as theTCRα chain, the TCRβ chain, the tumor-specific TCR, or thetumor-specific TCR construct). In some embodiments, there is provided anexpression vector encoding the tumor-specific TCR or components orderivatives thereof (such as the TCRα chain, the TCRβ chain, thetumor-specific TCR, or the tumor-specific TCR construct). In someembodiments, there is provided an isolated host cell expressing thetumor-specific TCR or components or derivatives thereof (such as theTCRα chain, the TCRβ chain, the tumor-specific TCR, or thetumor-specific TCR construct).

Nucleic acid sequences encoding exemplary tumor-specific TCRs are shownin Table 1 and FIGS. 25A-25B. In some embodiments, there is provided anisolated nucleic acid encoding a TCRα comprising a nucleic acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to any one ofthe nucleic acid sequences of SEQ ID NOs: 6, 12, 18, 24, 30, 36, 42, 48,54, 60, 66, 72, 78, 89, and 95. In some embodiments, there is providedan isolated nucleic acid encoding a TCRβ comprising a nucleic acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to any one of the nucleic acid sequences of SEQ ID NOs: 9, 15, 21, 27,33, 39, 45, 51, 57, 63, 69, 75, 81, 92 and 98.

In some embodiments, there is provided one or more vectors comprising:(a) a TCRα chain-encoding nucleic acid comprising a nucleic acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to any one of the nucleic acid sequences of SEQ ID NOs: 6, 12 and 18,and a TCRβ chain-encoding nucleic acid comprising a nucleic acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to any one of the nucleic acid sequences of SEQ ID NOs: 9, 15 and 21;(b) a TCRα chain-encoding nucleic acid comprising a nucleic acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to any one of the nucleic acid sequences of SEQ ID NOs: 24, 30, 36, 42,48 and 54, and a TCRβ chain-encoding nucleic acid comprising a nucleicacid sequence having at least about 80% identity (e.g., at least aboutany one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to any one of the nucleic acid sequences of SEQ ID NOs: 27,33, 39, 45, 51 and 57; or (c) a TCRα chain-encoding nucleic acidcomprising a nucleic acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to any one of the nucleic acid sequences of SEQID NOs: 60, 66, 72, 78, 89 and 95, and a TCRβ chain-encoding nucleicacid comprising a nucleic acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to any one of the nucleic acidsequences of SEQ ID NOs: 63, 69, 75, 81, 92 and 98.

In some embodiments, there is provided one or more vectors comprising:(a) a TCRα chain-encoding nucleic acid comprising a nucleic acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 6; a TCRβ chain-encoding nucleic acid comprising a nucleicacid sequence having at least about 80% identity (e.g., at least aboutany one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 9; (b) a TCRα chain-encoding nucleic acidcomprising a nucleic acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 12; a TCRβ chain-encoding nucleicacid comprising a nucleic acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 15; (c) a TCRαchain-encoding nucleic acid comprising a nucleic acid sequence having atleast about 80% identity (e.g., at least about any one of 85%, 90%, 95%,98% or higher identity, or having 100% identity) to SEQ ID NO: 18; aTCRβ chain-encoding nucleic acid comprising a nucleic acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:21; (d) a TCRα chain-encoding nucleic acid comprising a nucleic acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 24; a TCRβ chain-encoding nucleic acid comprising anucleic acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 27; (e) a TCRα chain-encoding nucleic acidcomprising a nucleic acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 30; a TCRβ chain-encoding nucleicacid comprising a nucleic acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 33; (f) a TCRαchain-encoding nucleic acid comprising a nucleic acid sequence having atleast about 80% identity (e.g., at least about any one of 85%, 90%, 95%,98% or higher identity, or having 100% identity) to SEQ ID NO: 36; aTCRβ chain-encoding nucleic acid comprising a nucleic acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:39; (g) a TCRα chain-encoding nucleic acid comprising a nucleic acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 42; a TCRβ chain-encoding nucleic acid comprising anucleic acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 45; (h) a TCRα chain-encoding nucleic acidcomprising a nucleic acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 48; a TCRβ chain-encoding nucleicacid comprising a nucleic acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 51; (i) a TCRαchain-encoding nucleic acid comprising a nucleic acid sequence having atleast about 80% identity (e.g., at least about any one of 85%, 90%, 95%,98% or higher identity, or having 100% identity) to SEQ ID NO: 54; aTCRβ chain-encoding nucleic acid comprising a nucleic acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:57; (j) a TCRα chain-encoding nucleic acid comprising a nucleic acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 60; a TCRβ chain-encoding nucleic acid comprising anucleic acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 63; (k) a TCRα chain-encoding nucleic acidcomprising a nucleic acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 66; a TCRβ chain-encoding nucleicacid comprising a nucleic acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 69; (1) a TCRαchain-encoding nucleic acid comprising a nucleic acid sequence having atleast about 80% identity (e.g., at least about any one of 85%, 90%, 95%,98% or higher identity, or having 100% identity) to SEQ ID NO: 72; aTCRβ chain-encoding nucleic acid comprising a nucleic acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:75; (m) a TCRα chain-encoding nucleic acid comprising a nucleic acidsequence having at least about 80% identity (e.g., at least about anyone of 85%, 90%, 95%, 98% or higher identity, or having 100% identity)to SEQ ID NO: 78; a TCRβ chain-encoding nucleic acid comprising anucleic acid sequence having at least about 80% identity (e.g., at leastabout any one of 85%, 90%, 95%, 98% or higher identity, or having 100%identity) to SEQ ID NO: 81; (n) a TCRα chain-encoding nucleic acidcomprising a nucleic acid sequence having at least about 80% identity(e.g., at least about any one of 85%, 90%, 95%, 98% or higher identity,or having 100% identity) to SEQ ID NO: 89; a TCRβ chain-encoding nucleicacid comprising a nucleic acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to SEQ ID NO: 92; or (o) a TCRαchain-encoding nucleic acid comprising a nucleic acid sequence having atleast about 80% identity (e.g., at least about any one of 85%, 90%, 95%,98% or higher identity, or having 100% identity) to SEQ ID NO: 95; aTCRβ chain-encoding nucleic acid comprising a nucleic acid sequencehaving at least about 80% identity (e.g., at least about any one of 85%,90%, 95%, 98% or higher identity, or having 100% identity) to SEQ ID NO:98.

In some embodiments, there is provided an isolated nucleic acid (such asa vector) comprising a tumor-specific TCR construct, wherein the nucleicacid comprises a nucleic acid sequence having at least about 80%identity (e.g., at least about any one of 85%, 90%, 95%, 98% or higheridentity, or having 100% identity) to any one of the nucleic acidsequences selected from the group consisting of SEQ ID NOs: 144, 146,148, 150, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192 and 194.

In brief summary, the expression of an tumor-specific TCR by a nucleicacid encoding the tumor-specific TCR can be achieved by inserting thenucleic acid into an appropriate expression vector, such that thenucleic acid is operably linked to 5′ and 3′ regulatory elements,including for example a promoter (e.g., a lymphocyte-specific promoter)and a 3′ untranslated region (UTR). The vectors can be suitable forreplication and integration in eukaryotic host cells. Typical cloningand expression vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence.

The nucleic acids of the present invention may also be used for nucleicacid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In some embodiments, the inventionprovides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to, a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers (see, e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In someembodiments, lentivirus vectors are used. Vectors derived fromretroviruses such as the lentivirus are suitable tools to achievelong-term gene transfer since they allow long-term, stable integrationof a transgene and its propagation in daughter cells. Lentiviral vectorshave the added advantage over vectors derived from onco-retrovirusessuch as murine leukemia viruses in that they can transducenon-proliferating cells. They also have the added advantage of lowimmunogenicity.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-la(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter.

Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Exemplary induciblepromoter systems for use in eukaryotic cells include, but are notlimited to, hormone-regulated elements (e.g., see Mader, S. and White,J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), syntheticligandregulated elements (see, e.g., Spencer, D. M. et al 1993) Science262: 1019-1024) and ionizing radiation-regulated elements (e.g., seeManome, Y. et al. (1993) Biochemistry 32: 10607-10613; Datta, R. et al.(1992) Proc. Natl. Acad. Sci. USA 89: 1014- 10153). Further exemplaryinducible promoter systems for use in in vitro or in vivo mammaliansystems are reviewed in Gingrich et al. (1998) Annual Rev. Neurosci21:377-405.

In order to assess the expression of a polypeptide or portions thereof,the expression vector to be introduced into a cell can also containeither a selectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother aspects, the selectable marker may be carried on a separate pieceof DNA and used in a cotransfection procedure. Both selectable markersand reporter genes may be flanked with appropriate regulatory sequencesto enable expression in the host cells. Useful selectable markersinclude, for example, antibiotic-resistance genes, such as neo and thelike.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,β-galactosidase, chloramphenicol acetyl transferase, secreted alkalinephosphatase, or the green fluorescent protein gene (e.g., Ui-Tel et al.,2000 FEBS Letters 479: 79-82). Suitable expression systems are wellknown and may be prepared using known techniques or obtainedcommercially. In general, the construct with the minimal 5′ flankingregion showing the highest level of expression of reporter gene isidentified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In some embodiments, there is provided nucleic acid encoding atumor-specific TCR according to any of the tumor-specific TCRs describedherein. In some embodiments, the nucleic acid encoding thetumor-specific TCR comprises a first nucleic acid sequence encoding theTCRα chain of the tumor-specific TCR and a second nucleic acid sequenceencoding the TCRβ chain of the tumor-specific TCR. In some embodiments,the first nucleic acid sequence is located on a first vector and thesecond nucleic acid sequence is located on a second vector. In someembodiments, the first and second nucleic acid sequences are located onthe same vector. In some embodiments, the first nucleic acid sequence isfused to the second nucleic acid sequence via a third nucleic acidsequence encoding a self-cleavable linker, such as P2A, T2A, E2A, or F2Apeptide. Vectors may be selected, for example, from the group consistingof mammalian expression vectors and viral vectors (such as those derivedfrom retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses). In some embodiments, the first nucleic acidsequence is under the control of a first promoter and the second nucleicacid sequence is under the control of a second promoter. In someembodiments, the first and second promoters have the same sequence. Insome embodiments, the first and second promoters have differentsequences. In some embodiments, the first and second nucleic acidsequences are expressed as a single transcript under the control of asingle promoter in a multicistronic (such as a bicistronic) vector. Seefor example Kim, JH, et al., PLoS One 6(4):e18556, 2011. In someembodiments, the first, second, and/or single promoters are inducible.

Engineered Immune Cells

In some embodiments, there is provided an engineered immune cell (suchas a T cell) expressing a tumor-specific TCR according to any of thetumor-specific TCRs described herein. In some embodiments, theengineered immune cell comprises a nucleic acid encoding thetumor-specific TCR, wherein the tumor-specific TCR is expressed from thenucleic acid and localized to the surface of the engineered immune cell.In some embodiments, the engineered immune cell is a T cell. In someembodiments, the engineered immune cell is selected from the groupconsisting of a PBMC, a cytotoxic T cell, a helper T cell, a naturalkiller T cell, and a regulatory T cell. In some embodiments, theengineered immune cell does not express an endogenous TCR.

In some embodiments, there is provided an engineered T cell comprising atumor-specific TCRs according to any of the tumor-specific TCRs orcomponents or derivatives thereof (such as the TCRα chain, the TCRβchain, the tumor-specific TCR, or the tumor-specific TCR construct). Insome embodiments, the endogenous TCR of the engineered T cell is knockedout. In some embodiments, the engineered T cell is a TCR-T cell. In someembodiments, there is provided a pharmaceutical composition comprisingthe engineered T cell and a pharmaceutically acceptable excipient. Insome embodiments, the engineered T cell is derived from the individualreceiving the TCR-T treatment. In some embodiments, the engineered Tcell is derived from an allogenic individual.

In some embodiments, the engineered immune cell (e.g., T cell) expressesa plurality (such as about any one of 2, 3, 4, 5, 10, or more) oftumor-specific TCRs obtained using any one of the methods describedherein. In some embodiments, the engineered immune cell (e.g., T cell)expresses tumor-specific TCRs specifically recognizing a plurality oftarget tumor antigen peptides.

The engineered immune cells or pharmaceutical compositions thereof maybe useful for treating the individual from whom the tumor-specific TCRis obtained (e.g., as a maintenance therapy), or for treating anotherindividual, such as an allogenic individual, or an individual having thesame MHC genotype, HLA haplotype and/or expressing the same epitope onthe cancer cells.

Methods of Treatment Using Tumor-Specific TCRs

The present application provides cell-based immunotherapy methods oftreating cancer in an individual, comprising administering to theindividual an effective amount of an engineered immune cells (e.g., Tcells) expressing any one of the tumor-specific TCRs described herein.In some embodiments, the method is used as a maintenance therapy for aprevious MASCT received by the individual.

The methods described herein are suitable for treating various cancers,including liquid and solid cancers. In some embodiments, the cancer isselected from the group consisting of hepatocellular carcinoma, cervicalcancer, lung cancer, colorectal cancer, lymphoma, renal carcinoma,breast cancer, pancreatic cancer, gastric cancer, esophageal cancer,ovarian cancer, prostate cancer, nasopharyngeal carcinoma, melanoma,endometrial cancer, and brain cancer. The methods are applicable tocancers of all stages, including early stage, advanced stage andmetastatic cancer. For example, any one of the CEA-specific TCRs, theRGS5-specific TCRs and HPV 18-E7 TCRs described herein may be useful fortreating cervical cancer.

In some embodiments, the method reduces the severity of one or moresymptoms associated with the cancer by at least about any of 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to thecorresponding symptom in the same individual prior to treatment orcompared to the corresponding symptom in other individuals not receivingthe treatment method. In some embodiments, the method delays progressionof the cancer.

In some embodiments, the method is for treating hepatocellular carcinoma(HCC). In some embodiments, the HCC is early stage HCC, non-metastaticHCC, primary HCC, advanced HCC, locally advanced HCC, metastatic HCC,HCC in remission, or recurrent HCC. In some embodiments, the HCC islocalized resectable (i.e., tumors that are confined to a portion of theliver that allows for complete surgical removal), localized unresectable(i.e., the localized tumors may be unresectable because crucial bloodvessel structures are involved or because the liver is impaired), orunresectable (i.e., the tumors involve all lobes of the liver and/or hasspread to involve other organs (e.g., lung, lymph nodes, bone). In someembodiments, the HCC is, according to TNM classifications, a stage Itumor (single tumor without vascular invasion), a stage II tumor (singletumor with vascular invasion, or multiple tumors, none greater than 5cm), a stage III tumor (multiple tumors, any greater than 5 cm, ortumors involving major branch of portal or hepatic veins), a stage IVtumor (tumors with direct invasion of adjacent organs other than thegallbladder, or perforation of visceral peritoneum), N1 tumor (regionallymph node metastasis), or M1 tumor (distant metastasis). In someembodiments, the HCC is, according to AJCC (American Joint Commission onCancer) staging criteria, stage T1, T2, T3, or T4 HCC. In someembodiments, the HCC is any one of liver cell carcinomas, fibrolamellarvariants of HCC, and mixed hepatocellularcholangiocarcinomas. In someembodiments, the HCC is caused by Hepatitis B Virus (HBV) infection.

In some embodiments, the method is for treating lung cancer. In someembodiments, the lung cancer is a non-small cell lung cancer (NSCLC).Examples of NCSLC include, but are not limited to, large-cell carcinoma(e.g., large-cell neuroendocrine carcinoma, combined large-cellneuroendocrine carcinoma, basaloid carcinoma, lymphoepithelioma-likecarcinoma, clear cell carcinoma, and large-cell carcinoma with rhabdoidphenotype), adenocarcinoma (e.g., acinar, papillary (e.g.,bronchioloalveolar carcinoma, nonmucinous, mucinous, mixed mucinous andnonmucinous and indeterminate cell type), solid adenocarcinoma withmucin, adenocarcinoma with mixed subtypes, welldifferentiated fetaladenocarcinoma, mucinous (colloid) adenocarcinoma, mucinouscystadenocarcinoma, signet ring adenocarcinoma, and clear celladenocarcinoma), neuroendocrine lung tumors, and squamous cell carcinoma(e.g., papillary, clear cell, small cell, and basaloid). In someembodiments, the NSCLC may be, according to TNM classifications, a stageT tumor (primary tumor), a stage N tumor (regional lymph nodes), or astage M tumor (distant metastasis).

In some embodiments, the lung cancer is a carcinoid (typical oratypical), adenosquamous carcinoma, cylindroma, or carcinoma of thesalivary gland (e.g., adenoid cystic carcinoma or mucoepidermoidcarcinoma). In some embodiments, the lung cancer is a carcinoma withpleomorphic, sarcomatoid, or sarcomatous elements (e.g., carcinomas withspindle and/or giant cells, spindle cell carcinoma, giant cellcarcinoma, carcinosarcoma, or pulmonary blastoma). In some embodiments,the lung cancer is small cell lung cancer (SCLC; also called oat cellcarcinoma). The small cell lung cancer may be limited-stage, extensivestage or recurrent small cell lung cancer. In some embodiments, theindividual may be a human who has a gene, genetic mutation, orpolymorphism suspected or shown to be associated with lung cancer (e.g.,SASH1, LATS1, IGF2R, PARK2, KRAS, PTEN, Kras2, Krag, Pas1, ERCC1, XPD,IL8RA, EGFR, α₁-AD, EPHX, MMP1, MMP2, MMP3, MMP12, IL1 β, RAS, and/orAKT) or has one or more extra copies of a gene associated with lungcancer.

In some embodiments, the method is for treating cervical cancer. In someembodiments, the cervical cancer is early stage cervical cancer,non-metastatic cervical cancer, locally advanced cervical cancer,metastatic cervical cancer, cervical cancer in remission, unresectablecervical cancer, cervical cancer in an adjuvant setting, or cervicalcancer in a neoadjuvant setting. In some embodiments, the cervicalcancer is caused by human papillomavirus (HPV) infection. In someembodiments, the cervical cancer may be, according to TNMclassifications, a stage T tumor (primary tumor), a stage N tumor(regional lymph nodes), or a stage M tumor (distant metastasis). In someembodiments, the cervical cancer is any of stage 0, stage I (Tis, N0,M0), stage IA (T1a, N0, M0), stage IB (T1b, N0, M0), stage IIA (T2a, N0,M0), stage IIB (T2b, N0, M0), stage IIIA (T3a, N0, M0), stage IIIB (T3b,N0, M0, or T1-3, N1, M0) stage IVA (T4, N0, M0), or stage IVB (T1-T3,N0-N1, M1) cervical cancer. In some embodiments, the cervical cancer iscervical squamous cell carcinoma, cervical adenonocarcinoma, oradenosquamous carcinoma.

In some embodiments, the method is for treating breast cancer. In someembodiments, the breast cancer is early stage breast cancer,non-metastatic breast cancer, locally advanced breast cancer, metastaticbreast cancer, hormone receptor positive metastatic breast cancer,breast cancer in remission, breast cancer in an adjuvant setting, ductalcarcinoma in situ (DCIS), invasive ductal carcinoma (IDC), or breastcancer in a neoadjuvant setting. In some embodiments, the breast canceris hormone receptor positive metastatic breast cancer. In someembodiments, the breast cancer (which may be HER2 positive or HER2negative) is advanced breast cancer. In some embodiments, the breastcancer is ductal carcinoma in situ. In some embodiments, the individualmay be a human who has a gene, genetic mutation, or polymorphismassociated with breast cancer (e.g., BRCA1, BRCA2, ATM, CHEK2, RAD51,AR, DIRAS3, ERBB2, TP53, AKT, PTEN, and/or PI3K) or has one or moreextra copies of a gene (e.g., one or more extra copies of the HER2 gene)associated with breast cancer.

In some embodiments, the method is for treating pancreatic cancer. Insome embodiments, the pancreatic cancer includes, but is not limited to,serous microcystic adenoma, intraductal papillary mucinous neoplasm,mucinous cystic neoplasm, solid pseudopapillary neoplasm, pancreaticadenocarcinoma, pancreatic ductal carcinoma, or pancreatoblastoma. Insome embodiments, the pancreatic cancer is any of early stage pancreaticcancer, non-metastatic pancreatic cancer, primary pancreatic cancer,resected pancreatic cancer, advanced pancreatic cancer, locally advancedpancreatic cancer, metastatic pancreatic cancer, unresectable pancreaticcancer, pancreatic cancer in remission, recurrent pancreatic cancer,pancreatic cancer in an adjuvant setting, or pancreatic cancer in aneoadjuvant setting.

In some embodiments, the method is for treating ovarian cancer. In someembodiments, the ovarian cancer is ovarian epithelial cancer. Exemplaryovarian epithelial cancer histological classifications include: serouscystomas (e.g., serous benign cystadenomas, serous cystadenomas withproliferating activity of the epithelial cells and nuclear abnormalitiesbut with no infiltrative destructive growth, or serouscystadenocarcinomas), mucinous cystomas (e.g., mucinous benigncystadenomas, mucinous cystadenomas with proliferating activity of theepithelial cells and nuclear abnormalities but with no infiltrativedestructive growth, or mucinous cystadenocarcinomas), endometrioidtumors (e.g., endometrioid benign cysts, endometrioid tumors withproliferating activity of the epithelial cells and nuclear abnormalitiesbut with no infiltrative destructive growth, or endometrioidadenocarcinomas), clear cell (mesonephroid) tumors (e.g., benign clearcell tumors, clear cell tumors with proliferating activity of theepithelial cells and nuclear abnormalities but with no infiltrativedestructive growth, or clear cell cystadenocarcinomas), unclassifiedtumors that cannot be allotted to one of the above groups, or othermalignant tumors. In various embodiments, the ovarian epithelial canceris stage I (e.g., stage IA, IB, or IC), stage II (e.g., stage IIA, IIB,or IIC), stage III (e.g., stage IIIA, IIIB, or IIIC), or stage IV. Insome embodiments, the individual may be a human who has a gene, geneticmutation, or polymorphism associated with ovarian cancer (e.g., BRCA1 orBRCA2) or has one or more extra copies of a gene associated with ovariancancer (e.g., one or more extra copies of the HER2 gene). In someembodiments, the ovarian cancer is an ovarian germ cell tumor. Exemplaryhistologic subtypes include dysgerminomas or other germ cell tumors(e.g., endodermal sinus tumors such as hepatoid or intestinal tumors,embryonal carcinomas, olyembryomas, choriocarcinomas, teratomas, ormixed form tumors). Exemplary teratomas are immature teratomas, matureteratomas, solid teratomas, and cystic teratomas (e.g., dermoid cystssuch as mature cystic teratomas, and dermoid cysts with malignanttransformation). Some teratomas are monodermal and highly specialized,such as struma ovarii, carcinoid, struma ovarii and carcinoid, or others(e.g., malignant neuroectodermal and ependymomas). In some embodiments,the ovarian germ cell tumor is stage I (e.g., stage IA, IB, or IC),stage II (e.g., stage IIA, IIB, or IIC), stage III (e.g., stage IIIA,IIIB, or IIIC), or stage IV.

Several viruses are related to cancer in humans. For example, HepatitisB virus (HBV) can cause chronic infection of the liver, increasing anindividual’s chance of liver cancer, or hepatocellular carcinoma (HCC).Human papilloma viruses (HPVs) are a group of more than 150 relatedviruses, which cause papilloma, or warts, when they infect and grow inskin or mucous membranes, such as the mouth, throat, or vagina. Severaltypes of HPV (including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,58, 59 and 6) are known to cause cervical cancer. HPVs also play a rolein inducing or causing other cancers of the genitalia, and are linked tosome cancers of the mouth and throat. Epstein-Barr virus (EBV) is a typeof herpes virus, which chronically infects and remains latent in Blymphocytes. EBV infection increases an individual’s risk of developingnasopharyngeal carcinoma and certain types of fast-growing lymphomassuch as Burkitt lymphoma. EBV is also linked to Hodgkin lymphoma andsome cases of gastric cancer. In addition to causing cancer orincreasing risk of developing cancer, viral infections, such asinfections with HBV, HPV, and EBV, may result in damage to tissues ororgans, which can increase the disease burden of an individual sufferingfrom a cancer, and contribute to cancer progression. It is known in theart that the human body can be induced to mount effective and specificimmune response, including cytotoxic T cell response, against severalcancer-related viruses, such as HBV, HPV and EBV, including theirvarious subtypes. Therefore, in some embodiments, there is provided amethod of treating a virus-related cancer in an individual, comprisingadministering to the individual an effective amount of an engineeredimmune cell (such as T cells) expressing any one of the tumor-specificTCRs described herein, wherein the target tumor antigen is derived fromthe virus. In some embodiments, the cancer is HBV-related hepatocellularcarcinoma, HPV-related cervical cancer, or EBV-related nasopharyngealcarcinoma.

The methods of treatment described herein can be used for any one ormore of the following purposes: alleviating one or more symptoms ofcancer, delaying progression of cancer, shrinking cancer tumor size,disrupting (such as destroying) cancer stroma, inhibiting cancer tumorgrowth, prolonging overall survival, prolonging disease-free survival,prolonging time to cancer disease progression, preventing or delayingcancer tumor metastasis, reducing (such as eradiating) preexistingcancer tumor metastasis, reducing incidence or burden of preexistingcancer tumor metastasis, preventing recurrence of cancer, and/orimproving clinical benefit of cancer.

In some embodiments, there is provided a method of inhibiting cancercell proliferation (such as tumor growth) in an individual, comprisingadministering to the individual an effective amount of an engineeredimmune cells (e.g., T cells) expressing any one of the tumor-specificTCRs described herein. In some embodiments, the method further comprisesadministering to the individual an effective amount of an effectiveamount of antigen-loaded DCs. In some embodiments, at least about 10%(including for example at least about any of 20%, 30%, 40%, 60%, 70%,80%, 90%, or 100%) cell proliferation is inhibited.

In some embodiments, there is provided a method of inhibiting tumormetastasis in an individual, comprising administering to the individualan effective amount of an engineered immune cells (e.g., T cells)expressing any one of the tumor-specific TCRs described herein. In someembodiments, the method further comprises administering to theindividual an effective amount of an effective amount of antigen-loadedDCs. In some embodiments, at least about 10% (including for example atleast about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%)metastasis is inhibited. In some embodiments, method of inhibitingmetastasis to lymph node is provided.

In some embodiments, there is provided a method of reducing tumor sizein an individual, comprising administering to the individual aneffective amount of an engineered immune cells (e.g., T cells)expressing any one of the tumor-specific TCRs described herein. In someembodiments, the method further comprises administering to theindividual an effective amount of an effective amount of antigen-loadedDCs. In some embodiments, the tumor size is reduced at least about 10%(including for example at least about any of 20%, 30%, 40%, 60%, 70%,80%, 90%, or 100%).

In some embodiments, there is provided a method of prolongingprogression-free survival of cancer in an individual, comprisingadministering to the individual an effective amount of an engineeredimmune cells (e.g., T cells) expressing any one of the tumor-specificTCRs described herein. In some embodiments, the method further comprisesadministering to the individual an effective amount of an effectiveamount of antigen-loaded DCs. In some embodiments, the method prolongsthe time to disease progression by at least any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 weeks.

In some embodiments, there is provided a method of prolonging survivalof an individual having cancer, comprising administering to theindividual an effective amount of an engineered immune cells (e.g., Tcells) expressing any one of the tumor-specific TCRs described herein.In some embodiments, the method further comprises administering to theindividual an effective amount of an effective amount of antigen-loadedDCs. In some embodiments, the method prolongs the time to diseaseprogression by at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 weeks. In some embodiments, the method prolongs the survivalof the individual by at least about any one of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 18, or 24 months.

In some embodiments, there is provided a method of reducing adverseeffects (AEs) and severe adverse effects (SAEs) in an individual havingcancer, comprising administering to the individual an effective amountof an engineered immune cells (e.g., T cells) expressing any one of thetumor-specific TCRs described herein. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofantigen-loaded DCs.

In some embodiments, the method is predictive of and/or results in anobjective response (such as a partial response or complete response). Insome embodiments, the method is predictive of and/or results in improvedquality of life.

Some cancer immunotherapies are associated with immune-related adverseevents (irAEs) in additional to common adverse events generallyassociated with other cancer therapies. IrAEs are usuallymechanistically related to either on-target T-cell toxicity againsttarget antigens that are expressed in normal, non-tumor tissue, socalled on-target off-tumor effect, or off-target effects such asbreaking of self-tolerance or epitope cross-reaction. IrAEs can lead tosevere symptoms and conditions on the dermatologic, gastrointestinal,endocrine, hepatic, ocular, neurologic, and other tissues or organs.Typical irAEs reported for cancer immunotherapy methods known in the artinclude fatal immune-mediated dermatitis, pneumonia, colitis,lymphocytic hypophysitis, pancreatitis, lymphadenopathy, endocrinedisorders, CNS toxicity, and the like. In some embodiments, thetreatment method is associated with low incidence of adverse events,such as irAEs. In some embodiments, less than about any one of 50%, 40%,30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of individuals experience irAEs,such as irAEs of Grade 2-5.

Generally, dosages, schedules, and routes of administration of theengineered immune cell (such as T cells) expressing any one of thetumor-specific TCRs described herein may be determined according to thesize and condition of the individual, and according to standardpharmaceutical practice. Exemplary routes of administration includeintravenous, intra-arterial, intraperitoneal, intrapulmonary,intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, or transdermal. In some embodiments, theengineered immune cell (such as T cells) expressing the tumor-specificTCR is administered intravenously.

The dose of the cells administered to an individual may vary accordingto, for example, the particular type of cells being administered, theroute of administration, and the particular type and stage of cancerbeing treated. The amount should be sufficient to produce a desirableresponse, such as a therapeutic response against cancer, but withoutsevere toxicity or adverse events. In some embodiments, the amount ofthe engineered immune cell (such as T cells) expressing thetumor-specific TCR to be administered is a therapeutically effectiveamount. In some embodiments, the amount of the cells is an amountsufficient to decrease the size of a tumor, decrease the number ofcancer cells, or decrease the growth rate of a tumor by at least aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% comparedto the corresponding tumor size, number of cancer cells, or tumor growthrate in the same individual prior to treatment or compared to thecorresponding activity in other individuals not receiving the treatment.Standard methods can be used to measure the magnitude of this effect,such as in vitro assays with purified enzyme, cell-based assays, animalmodels, or human testing.

In some embodiments, a stabilizing agent or an excipient, such as humanalbumin, is used together with the engineered immune cell expressing thetumor-specific TCR(s).

The treatment method may comprise a single treatment, or repeatedtreatments. In some embodiments, the engineered immune cell expressingthe tumor-specific TCR is administered for at least about any one of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In some embodiments,the engineered immune cell expressing the tumor-specific TCR isadministered at least 3 times. In some embodiments, the treatment methodis repeated once per week, once 2 weeks, once 3 weeks, once 4 weeks,once per month, once per 2 months, once per 3 months, once per 4 months,once per 5 months, once per 6 months, once per 7 months, once per 8months, once per 9 months, or once per year.

The treatment method provided herein may be used as a first therapy,second therapy, third therapy, or combination therapy with other typesof cancer therapies known in the art, such as chemotherapy, surgery,radiation, gene therapy, immunotherapy, bone marrow transplantation,stem cell transplantation, targeted therapy, cryotherapy, ultrasoundtherapy, photodynamic therapy, radio-frequency ablation or the like, inan adjuvant setting or a neoadjuvant setting. In some embodiments, thetreatment method is used as a first therapy. In some embodiments, thereexists no other approved anti-cancer therapy for the individual. In someembodiments, the treatment method is used as a second therapy, whereinthe individual has previously received resection, radio-frequencyablation, chemotherapy, radiation therapy, or other types of cancertherapy. In some embodiments, the individual has progressed or has notbeen able to tolerate standard anti-cancer therapy. In some embodiments,the individual receives other types of cancer therapy prior to,concurrently with, or after receiving the treatment method describedherein. For example, the treatment method described herein may precedeor follow the other cancer therapy (such as chemotherapy, radiation,surgery or combination thereof) by intervals ranging from minutes, days,weeks to months. In some embodiments, the interval between the first andthe second therapy is such that the engineered immune cell (e.g., Tcell) expressing the tumor-specific TCR is and the other cancer therapy(such as chemotherapy, radiation, surgery, or combination thereof) wouldbe able to exert an advantageously combined effect on the individual.Additionally, a person having a greater risk of developing aproliferative disease may receive treatments to inhibit and/or delay thedevelopment of the disease.

V. Compositions, Kits and Articles of Manufacture

The present application further provides kits, compositions (such aspharmaceutical compositions), and articles of manufacture for use in anyembodiment of the methods of obtaining a plurality of TCRs recognizing atarget tumor antigen peptide and methods of treatment described herein.

In some embodiments, there is provided a kit useful for cancerimmunotherapy, comprising at least 10 tumor antigen peptides. A personskilled in the art may use any combinations of tumor antigen peptidesfrom the first core group and optionally any combinations of cancer-typespecific antigen peptides from the second group, and/or neoantigenpeptides to load a population of DCs, which can further be used toprepare activated T cells for MASCT, tumor antigen-specific T cells orisolating tumor-specific TCRs for treating cancer in an individual.

In some embodiments, there is provided a kit comprising any one of thetumor-specific TCRs described herein, or nucleic acid(s) or vectorencoding the tumor-specific TCR thereof. In some embodiments, there isprovided a kit comprising a library of tumor-specific TCRs obtainedusing any one of the methods described herein with T cells or PBMCs froma plurality of individuals that have clinically benefitted from MASCT,nucleic acids encoding the tumor-specific TCRs thereof, or vectorsencoding the tumor-specific TCRs thereof. In some embodiments, thetumor-specific TCRs specifically recognize one or more tumor antigensselected from the group consisting of hTERT, p53, Survivin, NY-ESO-1,CEA, CCND1, RGS5, MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3,CDCA1, WT1, KRAS, PARP4, MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6,HPV18-E7, HPV58-E6, HPV58-E7, HBcAg, HBV polymerase, GPC3, SSX, and AFP.In some embodiments, the tumor-specific TCRs have HLA haplotyperestriction that is predominant in certain racial groups.

The kit may contain additional components, such as containers, reagents,culturing media, cytokines, immune checkpoint inhibitors, TLR agonists,buffers, antibodies, and the like to facilitate execution of anyembodiment of the treatment methods or cell preparation methodsdescribed herein. For example, in some embodiments, the kit furthercomprises a peripheral blood collection and storage apparatus, which canbe used to collect an individual’s peripheral blood. In someembodiments, the kit further comprises containers and reagents fordensity gradient centrifugation of peripheral blood, which can be usedto isolate PBMCs from a sample of human peripheral blood. In someembodiments, the kit further comprises culturing media, cytokines, orbuffers for obtaining DCs from peripheral blood. In some embodiments,the kit further comprises culturing media, TLR agonists (e.g., MPLA),IFNy, PGE2, reagents and buffers for loading the plurality of tumorantigen peptides into DCs. In some embodiments, the kit furthercomprises cytokines (e.g., IL-2, IL-7, IL-15 and IL-21), immunecheckpoint inhibitors (e.g., anti-PD1 antibody), anti-CD3 antibody(e.g., OKT-3), buffers, or culturing media for co-culturing T cells,enriched activated T cells, or tumor antigen-specific T cells withantigen-loaded APCs (e.g., DCs). In some embodiments, the kit furthercomprises cell line APCs, such as LCL cells. In some embodiments, thekit further comprises antibodies, magnetic beads, and columns forenriching activated T cells expressing a cytokine (e.g., IFNy). In someembodiments, the kit further comprises containers, buffers, and reagentsfor freezing and storing PBMCs or T cells. In some embodiments, the kitfurther comprises reagents for determining the mutation load (such as inone or more MHC genes) in cancer cells. In some embodiments, the kitfurther comprises an immune checkpoint inhibitor for combination therapywith the treatment method. In some embodiments, the kit furthercomprises reagents for identifying a neoantigen (such as by sequencing)in a tumor sample. In some embodiments, the kit further comprises anELISPOT assay for assessing specific immune response against one or moretumor antigen peptides. In some embodiments, the kit further comprisesprimers and reagents for amplifying TCR genes, and/or next-generationsequencing of TCR genes. In some embodiments, the kit further comprisesimmune cells, culturing medium and reagents for preparing engineeredimmune cells expressing the tumor-specific TCR(s).

The kits of the present application are in suitable packaging. Suitablepackaging include, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., Mylar or plastic bags), and the like. Kits mayoptionally provide additional components such as buffers andinterpretative information. The present application thus also providesarticles of manufacture, which include vials (such as sealed vials),bottles, jars, flexible packaging, and the like.

The instructions may also comprise instructions relating to the use ofthe tumor antigen peptides (and optionally additional componentsdescribed above). In some embodiments, the kit further comprises aninstructional manual, such as a manual describing a protocol of anembodiment of the treatment methods, or an embodiment of the cellpreparation methods as described herein. The instructions may alsoinclude information on dosage, dosing schedule, and routes ofadministration of the DCs, the activated T cells, or engineered immunecells expressing the tumor-specific TCR(s) prepared using the kit forthe intended treatment. In some embodiments, the kit further comprisesinstructions for selecting an individual for the treatment method. Insome embodiments, the kit further comprises instructions foradministering an immune checkpoint inhibitor in combination with thetreatment method, including, for example, information on dosage, dosingschedule, and route of administration of the immune checkpointinhibitor. In some embodiments, the kit further comprises instructionsfor identifying a neoantigen (such as by sequencing) in a tumor sample.In some embodiments, the kit further comprises instructions formonitoring an individual after receiving the treatment.

The containers may be unit doses, bulk packages (e.g., multi-dosepackages) or subunit doses. For example, kits may be provided thatcontain sufficient tumor antigen peptides as disclosed herein to preparesufficient tumor antigen-specific T cells, antigen-loaded APCs (such asDCs), and/or engineered immune cells expressing the tumor-specificTCR(s) to provide effective treatment of an individual for an extendedperiod, such as any of 3 weeks, 6 weeks, 9 weeks, 3 months, 4 months, 5months, 6 months, 8 months, 9 months, 1 year or more.

Kits may also include multiple unit doses of tumor antigen peptides ortumor-specific TCRs (or nucleic acids encoding tumor-specific TCRs) andinstructions for use and packaged in quantities sufficient for storageand use in pharmacies, for example, hospital pharmacies and compoundingpharmacies.

Further provided are kits, compositions (such as pharmaceuticalcompositions), and articles of manufacture of any one of the isolatedpopulation of cells (such as DCs, activated T cells, or engineeredimmune cells expressing tumor-specific TCRs) described herein.

The isolated population of cells (such as DCs, activated T cells, orengineered immune cells expressing tumor-specific TCRs) described hereinmay be used in pharmaceutical compositions or formulations, by combiningthe isolated population of cells described with a pharmaceuticallyacceptable carrier, excipients, stabilizing agents and/or other agents,which are known in the art, for use in the methods of treatment, methodsof administration, and dosage regimens described herein. In someembodiments, human albumin is used as a pharmaceutically acceptablecarrier.

Suitable pharmaceutical carriers include sterile water; saline,dextrose; dextrose in water or saline; condensation products of castoroil and ethylene oxide combining about 30 to about 35 moles of ethyleneoxide per mole of castor oil; liquid acid; lower alkanols; oils such ascorn oil; peanut oil, sesame oil and the like, with emulsifiers such asmono- or di-glyceride of a fatty acid, or a phosphatide, e.g., lecithin,and the like; glycols; polyalkylene glycols; aqueous media in thepresence of a suspending agent, for example, sodiumcarboxymethylcellulose; sodium alginate; poly(vinylpyrolidone) ; and thelike, alone, or with suitable dispensing agents such as lecithin;polyoxyethylene stearate; and the like. The carrier may also containadjuvants such as preserving stabilizing, wetting, emulsifying agentsand the like together with the penetration enhancer. The final form maybe sterile and may also be able to pass readily through an injectiondevice such as a hollow needle. The proper viscosity may be achieved andmaintained by the proper choice of solvents or excipients.

The pharmaceutical compositions described herein may include otheragents, excipients, or stabilizers to improve properties of thecomposition. Examples of suitable excipients and diluents include, butare not limited to, lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate and mineral oil. Insome embodiments, the pharmaceutical composition is formulated to have apH in the range of about 4.5 to about 9.0, including for example pHranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, orabout 6.5 to about 7.0. In some embodiments, the pharmaceuticalcomposition can also be made to be isotonic with blood by the additionof a suitable tonicity modifier, such as glycerol.

In some embodiments, the isolated cell composition (such as DCs,activated T cells, or engineered immune cells expressing tumor-specificTCRs) is suitable for administration to a human. In some embodiments,the compositions (such as pharmaceutical compositions) is suitable foradministration to a human by parenteral administration. Formulationssuitable for parenteral administration include aqueous and non-aqueous,isotonic sterile injection solutions, which can contain anti-oxidants,buffers, bacteriostats, and solutes that render the formulationcompatible with the blood of the intended recipient, and aqueous andnon-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, stabilizing agents, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in a conditionrequiring only the addition of the sterile liquid excipient methods oftreatment, methods of administration, and dosage regimens describedherein (i.e., water) for injection, immediately prior to use. In someembodiments, the compositions (such as pharmaceutical compositions) iscontained in a single-use vial, such as a single-use sealed vial. Insome embodiments, the composition (such as pharmaceutical composition)is contained in a multiuse vial. In some embodiments, the composition(such as pharmaceutical composition) is contained in bulk in acontainer.

Also provided are unit dosage forms comprising the isolated cellcompositions (such as pharmaceutical compositions) and formulationsdescribed herein. These unit dosage forms can be stored in a suitablepackaging in single or multiple unit dosages and may also be furthersterilized and sealed. In some embodiments, the composition (such aspharmaceutical composition) also includes one or more other compounds(or pharmaceutically acceptable salts thereof) that are useful fortreating cancer.

The present application further provides kits comprising any of theisolated population of cells, compositions (such as pharmaceuticalcompositions), formulations, unit dosages, and articles of manufacturedescribed herein for use in the methods of treatment, methods ofadministration, and dosage regimens described herein.

VI. Exemplary Embodiments

Among the embodiments provided herein are:

-   1. A method of obtaining a plurality of T cell receptors (TCRs)    specifically recognizing a target tumor antigen peptide, comprising:    -   a) a first co-culturing step comprising co-culturing a first        population of dendritic cells (DCs) loaded with the target tumor        antigen peptide with a population of T cells from an individual        to obtain a first co-culture;    -   b) an enrichment step comprising subjecting the first co-culture        to an enrichment process to obtain enriched activated T cells;    -   c) a second co-culturing step comprising co-culturing the        enriched activated T cells with a second population of DCs        loaded with the target tumor antigen peptide to obtain a        population of tumor antigen-specific T cells, wherein at least        about 10% of the tumor antigen-specific T cells specifically        responds to the target tumor antigen peptide; and    -   d) a sequencing step, comprising subjecting the tumor        antigen-specific T cells to next-generation sequencing to        identify a plurality of pairs of genes encoding TCRα and TCRβ,        thereby providing the plurality of T cell receptors based on        paired genes encoding TCRα and TCRβ;        -   wherein the individual has clinically benefitted from a            Multiple Antigen Specific Cell Therapy (MASCT) comprising            administering to the individual an effective amount of            activated T cells prepared by co-culturing a population of T            cells with a population of dendritic cells loaded with a            plurality of tumor antigen peptides comprising the target            tumor antigen peptide.-   2. The method of embodiment 1, wherein the first co-culturing step    is carried out for about 1 to about 3 days prior to the enrichment    step.-   3. The method of embodiment 1 or 2, wherein the ratio between the    population of T cells to the first population of DCs loaded with the    target tumor antigen peptide is no more than about 30:1.-   4. The method of any one of embodiments 1-3, wherein the population    of T cells in the first co-culturing step is present in PBMCs.-   5. The method of any one of embodiments 1-4, wherein the first    population of DCs loaded with the target tumor antigen peptide and    the population of T cells are co-cultured in a first co-culture    medium comprising one or more cytokines (e.g., IL-2 or a plurality    of cytokines) and an immune checkpoint inhibitor.-   6. The method of embodiment 5, wherein the first co-culture medium    comprises IL-2, IL-7, IL-15 and IL-21 and an anti-PD-1 antibody.-   7. The method of embodiment 5, wherein the first co-culture medium    comprises IL-2 and an anti-PD-1 antibody.-   8. The method of any one of embodiments 1-7, wherein the enrichment    step comprises contacting the first co-culture with antigen    presenting cells (APCs) loaded with the target tumor antigen peptide    to obtain a stimulated co-culture, and isolating from the stimulated    co-culture an enriched population of activated T cells using a    ligand that specifically recognizes a cytokine.-   9. The method of embodiment 8, wherein the cytokine is IFNy.-   10. The method of any one of embodiments 1-9, wherein the ratio    between the enriched population of activated T cells and the second    population of DCs loaded with the target tumor antigen peptide is    about 1:1 to about 20:1.-   11. The method of any one of embodiments 1-10, wherein the enriched    population of activated T cells and the second population of DCs    loaded with the target tumor antigen peptide are co-cultured for    about 12 to 25 days.-   12. The method of any one of embodiments 1-11, wherein the second    co-culturing step comprises co-culturing the second population of    DCs loaded with the target tumor antigen peptide with the enriched    population of activated T cells in an initial second co-culture    medium comprising an immune checkpoint inhibitor and optionally one    or more cytokines (e.g., IL-2 or a plurality of cytokines) to    provide a second co-culture; and adding an anti-CD3 antibody to the    second co-culture to obtain a population of tumor antigen-specific T    cells.-   13. The method of embodiment 12, wherein the anti-CD3 antibody is    added to the second co-culture no more than about 3 days (e.g., 2    days) after the second co-culturing step starts.-   14. The method of embodiment 12 or 13, wherein the anti-CD3 antibody    is OKT3.-   15. The method of any one of embodiments 12-14, wherein the initial    second co-culture medium comprises IL-2, IL-7, IL-15 and IL-21 and    an anti-PD-1 antibody.-   16. The method of any one of embodiments 12-14, wherein the second    co-culturing step comprises adding one or more cytokines to the    second co-culture.-   17. The method of embodiment 16, wherein the one or more cytokines    comprise IL-2.-   18. The method of embodiment 16 or 17, wherein the one or more    cytokines is added to the second co-culture no more than about 3    days (e.g., about 2 days) after the second co-culturing step starts.-   19. The method of any one of embodiments 12-18, wherein the initial    second co-culture medium comprises IL-2 and an anti-PD-1 antibody.-   20. The method of any one of embodiments 1-19, further comprising a    third co-culturing step comprising co-culturing a population of the    tumor antigen-specific T cells with a population of antigen    presenting cells (APCs) loaded with target tumor antigen peptide to    obtain a second population of tumor antigen-specific T cells,    wherein the second population of tumor antigen-specific T cells are    subjected to next-generation sequencing in the sequencing step.-   21. The method of embodiment 20, wherein the APCs are PBMCs, DCs, or    cell line APCs.-   22. The method of embodiment 20 or 21, wherein the ratio between the    population of tumor antigen-specific T cells and the population of    APCs loaded with the target tumor antigen peptide is about 1:1 to    about 20:1.-   23. The method of any one of embodiments 20-22, wherein the    population of tumor antigen-specific T cells and the population of    APCs loaded with the target tumor antigen peptide are co-cultured    for about 5 to 9 days.-   24. The method of any one of embodiments 20-23, wherein the    population of tumor antigen-specific T cells and the population of    APCs loaded with the target tumor antigen peptide are co-cultured in    a third co-culture medium comprising one or more cytokines (e.g., a    plurality of cytokines) and an anti-CD3 antibody.-   25. The method of embodiment 24, wherein the third co-culture medium    comprises IL-2 and OKT3.-   26. The method of embodiment 24, wherein the third co-culture medium    comprises IL-2, IL-7, IL-15 and OKT3.-   27. The method of any one of embodiments 20-26, the third    co-culturing step is repeated.-   28. The method of any one of embodiments 1-27, wherein the target    tumor antigen peptide is derived from a tumor antigen selected from    the group consisting of hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1,    RGS5, MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3, CDCA1,    WT1, KRAS, PARP4, MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6,    HPV18-E7, HPV58-E6, HPV58-E7, HBcAg, HBV polymerase, GPC3, SSX, and    AFP.-   29. The method of embodiments 1-28, further comprising identifying a    target tumor epitope from the target tumor antigen peptide, wherein    the target tumor epitope elicits specific response by the enriched    population of activated T cells, and contacting a population of APCs    with the target tumor epitope to obtain the population of APCs    loaded with the target tumor antigen peptide.-   30. The method of any one of embodiments 1-29, wherein the    next-generation sequencing is single cell sequencing.-   31. The method of any one of embodiments 1-30, wherein the    individual has partial response (PR), complete response (CR), or    stable disease (SD) after receiving the MASCT; and/or wherein the    individual has tumor antigen-specific immune response(s).-   32. The method of any one of embodiments 1-31, wherein the MASCT    comprises: co-culturing a population of DCs loaded with a plurality    of tumor antigen peptides comprising the target tumor antigen    peptide and a population of T cells to obtain a population of    activated T cells.-   33. The method of any one of embodiments 1-32, wherein the MASCT    comprises:    -   (i) co-culturing a population of DCs loaded with a plurality of        tumor antigen peptides comprising the target tumor antigen        peptide and a population of T cells in an initial co-culture        medium comprising one or more cytokines (e.g., a plurality of        cytokines) and an immune checkpoint inhibitor to provide a        co-culture; and    -   (ii) adding an anti-CD3 antibody to the co-culture at about 3 to        7 days after the co-culturing starts, thereby obtaining the        population of activated T cells.-   34. The method of any one of embodiments 1-33, wherein the MASCT    comprises:    -   (i) contacting a population of DCs with a plurality of tumor        antigen peptides comprising the target tumor antigen peptide to        obtain a population of DCs loaded with the plurality of tumor        antigen peptides; and    -   (ii) culturing the population of DCs loaded with the plurality        of tumor antigen peptides in a DC maturation medium comprising        MPLA.-   35. The method of embodiment 34, wherein the DC maturation medium    comprises INFy, MPLA and PGE2.-   36. The method of any one of embodiments 1-35, wherein the MASCT    comprises administering to the individual an effective amount of the    DCs loaded with the plurality of tumor antigen peptides.-   37. The method of any one of embodiments 1-36, wherein the    individual has previously received the MASCT for at least three    times.-   38. The method of any one of embodiments 1-37, wherein the tumor    antigen-specific T cells are stimulated with APCs loaded with the    target tumor antigen peptide prior to the next-generation    sequencing.-   39. The method of any one of embodiments 1-38, wherein TCRs    specifically recognizing a plurality of target tumor antigen    peptides are obtained in parallel.-   40. The method of any one of embodiments 1-39, further comprising    expressing each pair of genes encoding TCRα and TCRβ in a host    immune cell to provide an engineered immune cell expressing a TCR,    and assessing response of the engineered immune cell to the target    tumor antigen peptide.-   41. A method of obtaining a TCR specifically recognizing a target    tumor antigen peptide, comprising the method of embodiment 40,    wherein the TCR is selected based on the response of the engineered    immune cell expressing the TCR to the target tumor antigen peptide.-   42. The method of embodiment 41, further comprising determining HLA    restriction of the TCR.-   43. The method of embodiment 42, wherein the TCR has a HLA haplotype    restriction that is predominant in Asians.-   44. The method of any one of embodiments 41-43, further comprising    affinity maturation of the TCR.-   45. The method of any one of embodiments 41-44, further comprising    enhancing the paring of the TCRα and TCRβ chains in the TCR.-   46. The method of any one of embodiments 41-45, further comprising    enhancing the expression of the TCR.-   47. The method of any one of embodiments 41-46, wherein the target    tumor antigen peptide is derived from CEA, RSG-5 or HPV 18-E7.-   48. A tumor-specific TCR obtained using the method of any one of    embodiments 1-47.-   49. A tumor-specific TCR comprising:    -   (a) a TCRα chain comprising a complementary determining region        (CDR) 3 having at least about 90% sequence identity to any one        of the amino acid sequences of SEQ ID NOs: 4, 10, and 16; and a        TCRβ chain comprising a CDR3 comprising an amino acid sequence        having at least about 90% sequence identity to any one of the        amino acid sequences of SEQ ID NOs: 7, 13, and 19;    -   (b) a TCRα chain comprising a complementary determining region        (CDR) 3 having at least about 90% sequence identity to any one        of the amino acid sequences of SEQ ID NOs: 22, 28, 34, 40, 46,        and 52; and a TCRβ chain comprising a CDR3 comprising an amino        acid sequence having at least about 90% sequence identity to any        one of the amino acid sequences of SEQ ID NOs: 7, 13, 19, 25,        31, 37, 43, 49, and 55; or    -   (c) a TCRα chain comprising a complementary determining region        (CDR) 3 having at least about 90% sequence identity to any one        of the amino acid sequences of SEQ ID NOs: 58, 64, 70, 76, 87        and 93; and a TCRβ chain comprising a CDR3 comprising an amino        acid sequence having at least about 90% sequence identity to any        one of the amino acid sequences of SEQ ID NOs: 61, 67, 73, 79,        90 and 96.-   50. The tumor-specific TCR of embodiment 49, comprising:    -   (a) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 4, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 7;    -   (b) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 10, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 13;    -   (c) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 16, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 19;    -   (d) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 22, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 25;    -   (e) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 28, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 31;    -   (f) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 34, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 37;    -   (g) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 40, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 43;    -   (h) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 46, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 49;    -   (i) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 52, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 55;    -   (j) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 58, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 61;    -   (k) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 64, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 67;    -   (l) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 70, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 73;    -   (m)a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 76, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 79;    -   (n) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 87, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 90; or    -   (o) a TCRα chain comprising a CDR3 comprising the amino acid        sequence of SEQ ID NO: 93, and a TCRβ chain comprising a CDR3        comprising the amino acid sequence of SEQ ID NO: 96.-   51. A tumor-specific TCR comprising:    -   (a) a TCRα chain comprising CDRs of any one of the amino acid        sequences of SEQ ID NOs: 5, 11 and 17, and a TCRβ chain        comprising CDRs of any one of the amino acid sequences of SEQ ID        NOs: 8, 14 and 20;    -   (b) a TCRα chain comprising CDRs of any one of the amino acid        sequences of SEQ ID NOs: 23, 29, 35, 41, 47 and 53, and a TCRβ        chain comprising CDRs of any one of the amino acid sequences of        SEQ ID NOs: 26, 32, 38, 44, 50 and 56; or    -   (c) a TCRα chain comprising CDRs of any one of the amino acid        sequences of SEQ ID NOs: 59, 65, 71, 77, 88 and 94, and a TCRβ        chain comprising CDRs of any one of the amino acid sequences of        SEQ ID NOs: 62, 68, 74, 80, 91 and 97.-   52. The tumor-specific TCR of any one of embodiments 49-51,    comprising:    -   (a) a TCRα chain comprising an amino acid sequence having at        least about 80% identity to any one of the amino acid sequences        of SEQ ID NOs: 5, 11 and 17, and a TCRβ chain comprising an        amino acid sequence having at least about 80% identity to any        one of the amino acid sequences of SEQ ID NOs: 8, 14 and 20;    -   (b) a TCRα chain comprising an amino acid sequence having at        least about 80% identity to any one of the amino acid sequences        of SEQ ID NOs: 23, 29, 35, 41, 47 and 53, and a TCRβ chain        comprising an amino acid sequence having at least about 80%        identity to any one of the amino acid sequences of SEQ ID NOs:        26, 32, 38, 44, 50 and 56; or    -   (c) a TCRα chain comprising an amino acid sequence having at        least about 80% identity to any one of the amino acid sequences        of SEQ ID NOs: 59, 65, 71, 77, 88 and 94, and a TCRβ chain        comprising an amino acid sequence having at least about 80%        identity to any one of the amino acid sequences of SEQ ID NOs:        62, 68, 74, 80, 91 and 97.-   53. A tumor-specific TCR that specifically binds to an MHC/RGS5    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 254, a CDR2    comprising the amino acid sequence of SEQ ID NO: 255, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 28; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 256, a CDR2 comprising the amino acid sequence of SEQ ID NO:    257, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 31.-   54. The tumor-specific TCR of embodiment 53, wherein the RGS5    epitope comprises the amino acid sequence of SEQ ID NO: 82.-   55. The tumor-specific TCR of embodiment 53 or 54, wherein the MHC    is HLA-DPA1 *02:02/DPB1 *05:01.-   56. A tumor-specific TCR that specifically binds to an MHC/RGS5    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 258, a CDR2    comprising the amino acid sequence of SEQ ID NO: 259, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 34; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 260, a CDR2 comprising the amino acid sequence of SEQ ID NO:    261, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 37.-   57. The tumor-specific TCR of embodiment 56, wherein the RGS5    epitope comprises the amino acid sequence of SEQ ID NO: 82.-   58. The tumor-specific TCR of embodiment 56 or 57, wherein the MHC    is HLA-DRA/DRB 1*09:01 or HLA-DRA/DRB4*01:03.-   59. A tumor-specific TCR that specifically binds to an MHC/RGS5    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 262, a CDR2    comprising the amino acid sequence of SEQ ID NO: 263, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 40; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 264, a CDR2 comprising the amino acid sequence of SEQ ID NO:    265, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43.-   60. The tumor-specific TCR of embodiment 59, wherein the RGS5    epitope comprises the amino acid sequence of SEQ ID NO: 82.-   61. The tumor-specific TCR of embodiment 59 or 60, wherein the MHC    is HLA-DRA/DRB 1*09:01 or HLA-DRA/DRB4*01:03.-   62. A tumor-specific TCR that specifically binds to an MHC/RGS5    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 266, a CDR2    comprising the amino acid sequence of SEQ ID NO: 267, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 46; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 268, a CDR2 comprising the amino acid sequence of SEQ ID NO:    269, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 49.-   63. The tumor-specific TCR of embodiment 62, wherein the RGS5    epitope comprises the amino acid sequence of SEQ ID NO: 83.-   64. The tumor-specific TCR of embodiment 62 or 63, wherein the MHC    is HLA-DRA/DRB 1*09:01 or HLA-DRA/DRB4*01:03.-   65. A tumor-specific TCR that specifically binds to an MHC/RGS5    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 270, a CDR2    comprising the amino acid sequence of SEQ ID NO: 271, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 52; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 272, a CDR2 comprising the amino acid sequence of SEQ ID NO:    273, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 55.-   66. The tumor-specific TCR of embodiment 65, wherein the RGS5    epitope comprises the amino acid sequence of SEQ ID NO: 82.-   67. The tumor-specific TCR of embodiment 65 or 66, wherein the MHC    is HLA-DPA1 *02:02/DPB1 *05:01.-   68. A tumor-specific TCR that specifically binds to an MHC/HPV18-E7    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 274, a CDR2    comprising the amino acid sequence of SEQ ID NO: 275, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 58; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 276, a CDR2 comprising the amino acid sequence of SEQ ID NO:    277, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 61.-   69. The tumor-specific TCR of embodiment 68, wherein the HPV18-E7    epitope comprises the amino acid sequence of SEQ ID NO: 85.-   70. The tumor-specific TCR of embodiment 68 or 69, wherein the MHC    is HLA-DRA/DRB1 *09:01.-   71. A tumor-specific TCR that specifically binds to an MHC/HPV18-E7    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 278, a CDR2    comprising the amino acid sequence of SEQ ID NO: 279, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 70; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 280, a CDR2 comprising the amino acid sequence of SEQ ID NO:    281, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 73.-   72. The tumor-specific TCR of embodiment 71, wherein the HPV18-E7    epitope comprises the amino acid sequence of SEQ ID NO: 85.-   73. The tumor-specific TCR of embodiment 71 or 72, wherein the MHC    is HLA-DRA/DRB 1*09:01 or HLA-DRA/DRB4*01:03.-   74. A tumor-specific TCR that specifically binds to an MHC/HPV18-E7    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 282, a CDR2    comprising the amino acid sequence of SEQ ID NO: 283, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 76; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 284, a CDR2 comprising the amino acid sequence of SEQ ID NO:    285, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 79.-   75. The tumor-specific TCR of embodiment 74, wherein the HPV18-E7    epitope comprises the amino acid sequence of SEQ ID NO: 85.-   76. The tumor-specific TCR of embodiment 74 or 75, wherein the MHC    is HLA-II.-   77. A tumor-specific TCR that specifically binds to an MHC/HPV18-E7    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 286, a CDR2    comprising the amino acid sequence of SEQ ID NO: 287, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 87; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 288, a CDR2 comprising the amino acid sequence of SEQ ID NO:    289, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 90.-   78. The tumor-specific TCR of embodiment 77, wherein the HPV18-E7    epitope comprises the amino acid sequence of SEQ ID NO: 84.-   79. The tumor-specific TCR of embodiment 77 or 78, wherein the MHC    is HLA-DRA/DRB1 *09:01.-   80. A tumor-specific TCR that specifically binds to an MHC/HPV18-E7    epitope complex, comprising a TCRα chain comprising a CDR1    comprising the amino acid sequence of SEQ ID NO: 290, a CDR2    comprising the amino acid sequence of SEQ ID NO: 291, and a CDR3    comprising the amino acid sequence of SEQ ID NO: 93; and a TCRβ    chain comprising a CDR1 comprising the amino acid sequence of SEQ ID    NO: 292, a CDR2 comprising the amino acid sequence of SEQ ID NO:    293, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 96.-   81. The tumor-specific TCR of embodiment 80, wherein the HPV18-E7    epitope comprises the amino acid sequence of SEQ ID NO: 84.-   82. The tumor-specific TCR of embodiment 80 or 81, wherein the MHC    is HLA-DPA1*02:02/DPB1*05:01, HLA-DPA1*01:03/DPB1*02:01, or    HLA-DPA1*01:03/DPB1*05:01.-   83. The tumor-specific TCR of any one of embodiments 49-51 and    53-82, wherein the TCR is a human TCR.-   84. The tumor-specific TCR of any one of embodiments 49-51 and    53-82, wherein the TCR is a chimeric TCR, such as a murinized TCR,    e.g., a TCR comprising murine constant regions of TCRα and βchains.-   85. The tumor-specific TCR of any one of embodiments 49-51 and    53-82, wherein the TCR comprises murine TCR constant regions.-   86. An isolated nucleic acid encoding the TCRα chain and/or the TCRβ    chain of the tumor-specific TCR of any one of embodiments 48-85.-   87. A vector comprising the isolated nucleic acid of embodiment 86.-   88. An engineered immune cell comprising the tumor-specific TCR of    any one of embodiments 48-85, the isolated nucleic acid of    embodiment 86, or the vector of embodiment 87.-   89. The engineered immune cell of embodiment 88, wherein the immune    cell is a T cell.-   90. A pharmaceutical composition comprising the engineered immune    cell of embodiment 88 or 89, and a pharmaceutically acceptable    carrier.-   91. A method of treating a cancer in an individual, comprising    administering to the individual an effective amount of the    pharmaceutical composition of embodiment 90.-   92. A library of tumor-specific TCRs obtained using the method of    any one of embodiments 1-47.-   93. An epitope of RGS-5 comprising the amino acid sequence of SEQ ID    NO: 82 or 83.-   94. An epitope of HPV18-E7 comprising the amino acid sequence of SEQ    ID NO: 84, 85 or 86.-   95. An MHC/epitope complex comprising the epitope of embodiment 93    or 94 and an MHC molecule.

EXAMPLES

The examples below are intended to be purely exemplary of the presentapplication and should therefore not be considered to limit theinvention in any way. The following examples and detailed descriptionare offered by way of illustration and not by way of limitation.

Example 1: Specific Immune Response Against Tumor Antigen Peptides in aPatient Treated with MASCT

Patient WJ, female, was diagnosed with cervical cancer with vascularinvasion at age 41, and was tested positive with Human Papilloma Virus(HPV) DNA. She underwent curative resection, and a five-monthchemo-radio therapy. The patient took a second HPV DNA test, and wasconfirmed to be negative in serum HPV DNA. The clinical history andresponse of this patient is summarized in FIG. 2 .

About two years after the curative resection and chemo-radio therapy,the patient was diagnosed to have metastasis tumor on the rightsacroiliac joint bone according to Magnetic Resonance Imaging (MRI) andEmission Computed Tomography (ECT). The patient then received ten localradiotherapy treatments, followed by three MASCT treatment, administeredone per month. The MASCT treatment used PBMCs from the patient’s ownperipheral blood to prepare dendritic cells pulsed with a pool of 18antigen peptides, including a core group of 12 tumor-associated antigenpeptides, as well as a cervical cancer-specific group of 6 antigenpeptides derived from viral proteins of HPV. Briefly, monocytes from thepatient’s PBMCs were differentiated into immature DCs and then pulsedwith multiple synthetic peptide antigens including tumor-associatedantigens and HPV antigens. The immature DCs were further stimulated byTLR ligands to differentiate into mature DCs (mDCs). Half of mDCs weresubcutaneous injected to the patient. Maintaining T cells were preparedby culturing non-adherent PBMCs with anti-CD3 antibody (e.g., OKT3), andIL2. The other half of mDCs was co-cultured with the maintaining T cellsfor another 7-9 days before infusion. The patient was confirmed to haveHLA-A2 serotype (HLA-A0201⁺).

After four MASCT treatments, the patient’s ECT results showed that theright sacroiliac joint bone metastasis was reduced, and no newmetastasis was detected, indicating positive treatment outcome of MASCT.The patient received four additional MASCT treatments administered withan interval of about 1 month or 2 months. After a total of 8 MASCTtreatments, a sample of the patient’s PBMC was obtained and tested withan ELISPOT assay to determine whether the patient had a therapeuticallyeffective MHCrestricted T cell response to the antigen peptide pool andeach of the antigen peptides within the pool. The ELISPOT resultsdemonstrated enhanced T-cell response to the cervical carcinoma antigenpeptide pool, and individual antigen peptides within both the core groupof tumor-specific antigen peptides (such as hTERT, p53, CEA, and RGS5),and the cervical cancer-specific group of tumor antigen peptides (suchas HPV-3 and HPV-5). The patient’s ECT after a total of 8 MASCT showedfurther reduction of the right sacroiliac joint bone metastasis, and nonew metastasis sites, indicating that the MASCT treatment regimen wassuccessful in reducing tumor burden in the patient and in preventingtumor progression and further metastasis.

Based on the patient’s specific immune response, the antigen peptidepool was customized to provide a patient-specific antigen peptide poolby saving the responsive peptides that had induced specific responsesand removing the non-responsive peptides that did not induce specificresponses. The patient was further treated with four cycles of MASCTprepared using the patient-specific antigen peptide pool (referredherein as “precise MASCT”). After the four precise MASCT, The patient’sECT showed no development of the right sacroiliac joint bone metastasis,and no new metastasis sites.

The antigen peptide pool was further adjusted based on the specificimmune response of the patient, and the patient was treated with fourcycles of a 2^(nd) precise MASCT using the further adjusted peptideantigen pool. After the second four cycles of precise MASCT, the patientwas evaluated as having stable disease (SD). The patient-specificantigen peptide pool elicited enhanced specific responses asdemonstrated by the ELISPOT assay (FIG. 3A). In particular, HPV18-E7peptide, CEA peptide, and RGS5 peptide consistently yielded thestrongest specific response (FIG. 3B).

Adoptive transfer of tumor-specific TCR engineered T cells has showngreat efficacy against solid tumors. The clinical benefits of thispatient indicated that tumor-specific T cells were expanded in vivo andplayed an important role to control tumor progression. These T cells maybe the good sources to isolate tumor-specific TCRs.

Example 2: Identification of Paired Tumor Antigen Peptide-Specific TCRαand TCRβ Genes from Tumor Antigen-Specific T Cells

PBMC samples from the patient in Example 1 were obtained and used as thestarting material to prepare tumor antigen-specific T cells in thisexample. Twelve pairs of tumor antigen-specific TCRα and TCRβ genes (3pairs for CEA-specific TCRs, 3 pairs for RGS5-specific TCRs, and 6 pairsfor HPV18-E7-specific TCRs) were identified by next-generationsequencing of the tumor antigen-specific T cell samples.

Method 2 Cells Preparation

FIG. 4 provides an overview of the protocol of exemplary “Method 2”.Briefly, on Day 1, peripheral blood mononuclear cells (PBMCs) from thepatient were obtained by density gradient centrifugation on Lymphoprep(Nycomed Pharma, Oslo, Norway). The adherent monocytes were continued tobe cultured in AIM-V medium with 1000 U/mL GM-CSF and 500 U/mL IL-4 todifferentiate into immature dendritic cells (DCs). The resultingimmature DCs were pulsed with a peptide pool comprising three tumorantigen peptides derived from CEA, RGS5, and HPV18-E7 (1 µg/mL/peptide),and then cultured in a DC maturation medium to differentiate into matureDCs. On Day 8, PBMCs containing T cells were co-cultured withantigen-loaded mature DCs at a ratio between the T cells and the matureantigen-loaded DCs of about 15:1, and the co-culture medium contained acytokine cocktail and an anti-PD-1 antibody. On Day 11, the co-culturewas stimulated with PBMCs pulsed with the peptide pool. On Day 12, anIFNy secretion assay-cell enrichment and detection kit (Miltenyi Biotec)was used to isolate a population of IFNγ⁺ T cells. On Day 12, the IFNγ⁺T cells were co-cultured with antigen-loaded mature DCs at a ratiobetween the T cells and the antigen-loaded mature DCs of about 2:1 in amedium containing a cytokine cocktail, an anti-PD-1 antibody, and ananti-CD3 antibody from Day 12 to Day 25-35 to obtain tumorantigen-specific T cells.

Proliferation Assay

Cell proliferation was assessed using cell samples from Day 1 (PBMCs),Day 8 (start of co-culture), Day 11 (before IFNy enrichment and afterIFNy enrichment), and Days 17, 21, 25, 27, 31 and 32 (co-culture ofIFNγ⁺ T cells with antigen-loaded mature DCs). The numbers of cells ineach sample were counted.

As shown in FIG. 5 , the initial co-culture of antigen-loaded mature DCsand T cells yielded a small number of IFNγ⁺ T cells (Day 11). In theco-culture of enriched IFNγ⁺ T cells and antigen-loaded mature DCs, thenumber of cells continued to increase rapidly until Day 31, at whichtime point the total number of cells in the co-culture plateaued at morethan 10⁸. IFNγ production by tumor antigen-specific T cells

Various co-culture samples were each plated (T cells: 1 × 10⁶cells/well; PBMCs: 2.5× 10⁵ cells/well) in AIM-V medium and stimulatedwith 2 µg/mL of the peptide pool for 4 hours. The IFNy production levelsby tumor antigen-specific T cells in each sample were detected byintracellular cytokine staining and FACS analysis. Cell samplesincubated with 10 µg/mL irrelevant peptide were used as negativecontrols.

Antibodies for cell surface (e.g., anti-human CD3-FITC) or intracellularcytokine (e.g., anti-human IFNy-APC) staining were obtained from BDBiosciences. Intracellular cytokine staining was performed by fixing andpermeabilizing cells with cytofix/cytoperm (BD Biosciences). Flowcytometry was performed using FACS CantoII (BD Biosciences) flowcytometers and data was analyzed with the Flowjo program.

FIGS. 6A-6B show the percentages of tumor antigen-specific T cells inthe cell samples as determined by assessing IFNγ⁺ CD3⁺ cells in responseto stimulation by the tumor antigen peptide pool. After the enrichmentstep, the percentage of tumor antigen-specific T cells in the cellsample reached 83.5%. From Day 21 to Day 32, the co-cultures containedabout 10% tumor antigen-specific T cells that produced IFNy in responseto stimulation by the tumor antigen peptide pool. Non-specific T cellsthat produced IFNy in response to stimulation by irrelevant peptidesconstituted less than 1% in the co-cultures on Days 25-32.

Optimization of Method 2 (“Method 2m”) Cells Preparation

FIG. 7 provides an overview of the protocols of exemplary “Method 2m”.Briefly, on Day 1, peripheral blood mononuclear cells (PBMCs) from thepatient were obtained by density gradient centrifugation on Lymphoprep(Nycomed Pharma, Oslo, Norway). The adherent monocytes were continued tobe cultured in AIM-V medium with 1000 U/mL GM-CSF and 500 U/mL IL-4 todifferentiate into immature dendritic cells (DCs). The resultingimmature DCs were pulsed with a peptide pool comprising three tumorantigen peptides derived from CEA, RGS5, and HPV18-E7 (1 µg/mL/peptide),and then cultured in a DC maturation medium to differentiate into matureDCs. On Day 8, PBMCs containing T cells were co-cultured withantigen-loaded mature DCs at a ratio between the T cells and the matureantigen-loaded DCs of about 20:1, and the co-culture medium contained acytokine cocktail and an anti-PD-1 antibody. On Day 11, the co-culturewas stimulated with PBMCs pulsed with the peptide pool. On Day 12, anIFNy secretion assay-cell enrichment and detection kit (Miltenyi Biotec)was used to isolate a population of IFNγ⁺ T cells. Meanwhile, theantigen-loaded mature DCs were cultured in the DC maturation medium. OnDay 12, the IFNγ⁺ T cells were co-cultured with antigen-loaded matureDCs at a ratio between the T cells and the antigen-loaded mature DCs ofabout 1:1 in a medium containing a cytokine cocktail, an anti-PD-1antibody. On Day 13 or 14, an anti-CD3 antibody (OKT3) was added to theco-culture, which was continued to be cultured to Day 30 to obtain tumorantigen-specific T cells.

Proliferation Assay

Cell proliferation was assessed using cell samples from Day 1 (PBMCs),Day 9 (start of co-culture), Day 12 (before IFNy enrichment), Day 12(after IFNy enrichment), and Days 22 and 30 (co-culture of IFNγ⁺ T cellswith antigen-loaded mature DCs) as described above.

As shown in FIG. 8 , the initial co-culture of antigen-loaded mature DCsand T cells yielded a small number of IFNγ⁺ T cells (Day 12). In theco-culture of enriched IFNγ⁺ T cells and antigen-loaded mature DCs, thenumber of cells continued to increase rapidly until Day 31. The methodwith anti-CD3 antibody added on Day 14 resulted in a higher level ofcell proliferation.

IFNγ Production by Tumor Antigen-Specific T Cells

The IFNy production levels by tumor antigen-specific T cells in variousco-culture samples were determined as described above. FIGS. 9A-9B showthe percentages of tumor antigen-specific T cells in the cell samples asdetermined by assessing IFNγ⁺ CD3⁺ cells in response to stimulation bythe tumor antigen peptide pool. After the enrichment step, thepercentage of tumor antigen-specific T cells in the cell sample reached90.4%. From Day 22 to Day 30, the co-cultures contained about 6-10%tumor antigen-specific T cells that produced IFNy in response tostimulation by the tumor antigen peptide pool. The method with anti-CD3antibody added on Day 14 yielded a higher percentage of IFNγ⁺ CD3⁺cells. Consistent results were obtained by assessing IFNγ⁺ TNFα⁺ cells(FIG. 9C).

Screen of Subset of Tumor Antigen Peptide Pool

Five fragments of each of the three initial tumor antigen peptides, CEA,RGS5 and HPV18-E7, were designed and synthesized. Sub-pools of theinitial tumor antigen peptides and their fragments were preparedaccording to FIG. 13 . The tumor antigen-specific T cells obtained onDay 30 were stimulated with each of the sub-pool of tumor antigenpeptides, and the percentages of IFNγ⁺ CD3⁺ cells were determined. Asshown in FIGS. 14A-14B, Pool 2 and Pool 8 consistently yielded thehighest percentages of IFNγ⁺ CD3⁺ cells, which suggests that theRGS5-OLP5 fragment elicited the strongest specific response by the tumorantigen-specific T cells.

Next-Generation Sequencing of Tumor Antigen-Specific T Cells

Four samples of the tumor antigen-specific T cells prepared using Method2 were obtained and subjected to TCRα and TCRβ amplification coupled tonext-generation sequencing. The four samples are: (1) tumorantigen-specific T cells stimulated by the CEA peptide; (2) tumorantigen-specific T cells stimulated by the RGS5 peptide; (3) tumorantigen-specific T cells stimulated by the HPV18-E7 peptide; and (4)INFγ⁺CD3⁺ tumor antigen-specific T cells stimulated by the CEA peptideenriched by beads. As a control, a PBMC sample from the same patient wasalso subjected to the same next-generation sequencing analysis.

FIG. 10 shows flow cytometry results of tumor antigen-specific T cellsstimulated by the CEA, RGS5 and HPV18-E7 peptides respectively. Eachsample contained more than 90% of tumor antigen-specific T cells (acombination of CD8⁺ and CD4⁺ cells).

FIG. 11 shows the frequency of unique clonotypes of TCRα and TCRβsequences in the various samples. Tumor antigen-specific T cellsstimulated by specific tumor antigen peptides had increased frequency ofunique TCR clonotypes than tumor antigen-specific T cells stimulatedwith an irrelevant peptide. IFNy sorted tumor antigen-specific T cellsstimulated by tumor antigen peptides showed even higher frequency ofunique clonotypes of TCR.

Frozen PBMC samples obtained from the patient at three different timepoints (T1=June 2016, T2=September 2016, and T3=May 2017) were subjectedto the same tumor antigen-specific T cell preparation and sequencinganalysis. FIG. 12 shows the frequencies of unique TCRα and TCRβclonotypes identified from the bulk next-generation sequencing results.Certain TCRα and TCRβ genes were found in corresponding tumorantigen-specific T cell samples derived from two or all three frozenPBMC samples.

Single-cell TCRα and TCRβ amplification coupled to next-generationsequencing using the IPAIR™ technology (iRepertoire, Inc.) was appliedto the tumor antigen-specific T cell samples in order to obtain cognatepairing information of the TCRα and TCRβ genes. Three pairs ofCEA-specific TCRα and TCRβ genes (clones 1-3), three pairs ofRGS5-specific TCRα and TCRβ genes (clones 1-3), and six pairs ofHPV18-E7-specific TCRα and TCRβ genes (clones 1-6) were identified, andsynthesized. See Table 1 for sequence information of the exemplary TCRclones. Engineered T cells expressing each pair of TCRα and TCRβ genesare prepared, and the TCRs are validated by assessing tumorantigen-specific immune response by the engineered T cells using ELISPOTassay.

Comparison of Cytokine Cocktail v. IL-2 and Antigen Peptide Pool v.Single Antigen Peptide Cells Preparation

FIG. 20 provides an overview of protocols that compare addition ofcytokine cocktail v. IL-2 alone, and stimulation with DC loaded with apool of antigen peptides v. a single antigen peptide. Briefly, on Day 1,peripheral blood mononuclear cells (PBMCs) from the patient wereobtained by density gradient centrifugation on Lymphoprep (NycomedPharma, Oslo, Norway). The adherent monocytes were continued to becultured in AIM-V medium with 1000 U/mL GM-CSF and 500 U/mL IL-4 todifferentiate into immature dendritic cells (DCs). The resultingimmature DCs were pulsed with a peptide pool comprising three tumorantigen peptides derived from CEA, RGS5, and HPV18-E7 (1 µg/mL/peptide),and then cultured in a DC maturation medium to differentiate into matureDCs. On Day 9, PBMCs containing T cells were co-cultured withantigen-loaded mature DCs at a ratio between the T cells and the matureantigen-loaded DCs of about 15:1 to about 20:1, and the co-culturemedium contained a cytokine cocktail or IL-2 (no more than about 200IU/mL) and an anti-PD-1 antibody. On Day 11, the co-culture wasstimulated with PBMCs pulsed with the peptide pool or each individualpeptide. On Day 12, an IFNy secretion assay-cell enrichment anddetection kit (Miltenyi Biotec) was used to isolate a population ofIFNγ⁺ T cells. Meanwhile, the antigen-loaded mature DCs were cultured inthe DC maturation medium. The IFNγ⁺ T cells were co-cultured withantigen-loaded mature DCs at a ratio between the T cells and theantigen-loaded mature DCs of about 1:1 to about 3:1 in a mediumcontaining a cytokine cocktail added on Day 12 or IL-2 alone (at leastabout 2000 IU/mL) added on Day 14, and an anti-PD-1 antibody. On Day 14,an anti-CD3 antibody (OKT3) was added to the co-culture, which wascontinued to be cultured to Day 29-30 to obtain tumor antigen-specific Tcells.

Proliferation Assay and IFNγ Production by Tumor Antigen-Specific TCells

Cell proliferation was assessed using cell samples from Day 1 (PBMCs),Day 9 (start of co-culture), Day 12 (before IFNy enrichment), Day 12(after IFNy enrichment), and Days 19, 24 and 29 (co-culture of IFNγ⁺ Tcells with antigen-loaded mature DCs) by methods described in Example 2.

As shown in FIG. 21A, the initial co-culture of antigen-loaded matureDCs and T cells yielded a small number of IFNγ⁺ T cells (Day 12). In theco-culture of enriched IFNγ⁺ T cells and antigen-loaded mature DCs, thenumber of cells continued to increase rapidly until Day 29.

The IFNy production levels by tumor antigen-specific T cells in variousco-culture samples were determined by methods described in Example 2. Asshown in FIG. 21B, similar percentage of IFNγ⁺ T cells were obtainedafter the enrichment step on Day 12 with the cytokine cocktail or IL-2only added to the co-culture on Day 9.

Table 3 below compares the percentages of tumor-specific T cells in thecell samples on Day 19 (Test 1) and Day 29 (Test 2) as determined byassessing IFNγ⁺ CD3⁺and IFNγ⁺ TNFα⁺ cells in response to stimulation bythe tumor antigen peptide pool or individual antigen peptides. Protocolswith cytokine cocktail or IL-2 alone added on Day 9 and co-culture withDCs pulsed with tumor antigen pool or single tumor antigen yieldcomparable results in terms of T cell proliferation and percentages oftumor-specific T cells.

TABLE 3 Percentages of Tumor-specific T cells in Cell Samples Day 9 IL-2Tumor-specific T cells Test 1 Test 2 b a Pool CEA RGS5 HPV18E7 Pool CEARGS5 HPV18E7 CD3⁺ IFNγ⁺ (%) Pool 9.40 0.36 2.47 6.74 8.97 0.63 2.60 5.30CEA 13.90 0.27 6.69 5.46 6.66 0.70 3.64 3.29 RGS5 9.44 0.54 3.00 5.895.86 0.59 2.40 3.39 HPV18E7 12.65 0.50 4.44 6.05 7.16 1.31 2.50 4.39IFNγ+TNFα+ (%) Pool 4.15 0.16 1.08 4.07 5.32 0.52 2.40 3.94 CEA 8.960.21 4.25 4.07 3.76 0.64 1.85 2.69 RGS5 6.38 0.29 1.36 4.61 3.81 0.371.66 2.80 HPV18E7 7.90 0.44 2.85 3.65 4.69 0.91 1.92 2.90 Day 9 CocktailCD3⁺ IFNγ⁺ Pool 9.96 0.68 1.82 5.72 8.13 2.51 3.80 4.30 CEA 11.21 0.795.37 5.71 5.86 0.71 2.86 4.26 (%) RGS5 9.09 0.20 2.25 5.44 8.79 1.374.35 6.38 HPV18E7 12.08 1.16 6.09 4.16 9.73 1.87 4.93 3.83 IFNγ+TNFα+(%) Pool 5.33 0.56 0.89 3.54 4.07 1.06 1.91 2.42 CEA 6.96 0.62 3.51 3.672.84 0.69 0.42 2.71 RGS5 5.67 0.50 1.59 4.00 5.06 0.08 1.29 3.62 HPV18E76.48 0.69 3.84 2.30 4.63 0.62 2.56 2.52 a: stimulation/testingconditions b: culturing conditions

FIGS. 22A-22B compare T cell numbers and percentages of tumorantigen-specific T cells in various co-culture samples using protocolswith IL-2 or cytokine cocktail added on Days 9 and 12. The protocol withIL-2 added on Day 9, co-culture with DCs pulsed with the tumor antigenpeptide pool on Day 12 and IL-2 added on Day 14 yielded the highestpercentage of tumor-antigen-specific T cells on Days 19 and 29.

Example 3: Identification of Paired RGS5-Specific TCRα and TCRβ Genesfrom Tumor Antigen-Specific T Cells

A frozen stock of tumor antigen-specific T cells prepared using Method 2and Method 2m was used in this example to prepare a population of tumorantigen specific T cells with enhanced percentage (e.g., up to 50%) ofRGS5-specific T cells. Three pairs of RGS5-specific TCRα and TCRβ geneswere identified by single-cell sequencing of the tumor antigen-specificT cell samples.

Cells Preparation

FIGS. 15A-15B provide an overview of the protocols used in this example.Briefly, a sample of the co-culture containing tumor antigen-specific Tcells on Day 32 using Method 2 or on Day 30 using Method 2m described inExample 2 was frozen to provide a frozen stock of tumor antigen-specificT cells. On Day 1 of this experiment, a sample of the frozen stock oftumor antigen-specific T cells was thawed, and co-cultured with LCLcells (an APC cell line) loaded with RGS5-OLP5 (1 µg/mL) in a co-culturemedium comprising a cocktail of cytokines (IL-2, IL-7, IL-15), ananti-CD3 antibody (OKT-3) and RGS5-OLP5 until Day 9, with or withoutfeeder cells. The ratio between the tumor antigen-specific T cells andthe antigen-loaded LCL cells was about 4:1. The ratio between the tumorantigen-specific T cells, the feeder cells, and the antigen-loaded LCLcells was about 4:4:1. On Days 9 and 16, the cycles were repeated byco-culturing the tumor antigen-specific T cells with antigen-loaded LCLcells with or without the presence of feeder cells.

PBMCs and dendritic cells may be used in place of the LCL cells toprovide antigen-loaded APCs. The APCs may be loaded with a single tumorantigen peptide, an epitope fragment of a single tumor antigen peptide,a pool of tumor antigen peptides, or a pool of epitope fragments oftumor antigen peptides.

Proliferation Assay

Cell proliferation was assessed using cell samples from Days 1, 9, 16and 23 of the co-culture by methods described in Example 2.

As shown in FIG. 16 , cells continued to proliferate when a thawedpopulation of frozen tumor antigen-specific T cells was co-cultured withantigen-loaded LCL cells with or without feeder cells until Day 16. Thetotal cell numbers decreased by Day 23.

Cytokine Production by Tumor Antigen-Specific T Cells

The IFNy production levels by tumor antigen-specific T cells in variousco-culture samples were determined by methods described in Example 2.

FIG. 17A shows the percentages of tumor antigen-specific T cells in thecell samples as determined by assessing IFNγ⁺ CD3⁺ cells in response tostimulation by the RGS5-OLP5 peptide. On Day 16, the co-culture derivedfrom a frozen stock of tumor antigen-specific T cells using Method 2mcontained about 38.6% tumor antigen-specific T cells that produced IFNyin response to stimulation by the RGS5-OLP5 peptide. Notably, on Day 23,the co-culture derived from a frozen stock of tumor antigen-specific Tcells using Method 2m contained about 53.8% tumor antigen-specific Tcells that produced IFNy in response to stimulation by the RGS5-OLP5peptide. The co-cultures derived from a frozen stock of tumorantigen-specific T cells using Method 2 yielded lower percentages oftumor antigen-specific T cells that produced IFNy in response tostimulation by the RGS5-OLP5 peptide on Day 16 and Day 23. Consistentresults were obtained by assessing IFNγ⁺ TNFα⁺ cells (FIG. 17B). Theseresults suggest that repeated stimulation of the tumor antigen-specificT cells with APCs loaded with a tumor antigen peptide could enhancepercentage of T cells that specifically respond to the tumor antigenpeptide.

Next-Generation Sequencing of Tumor Antigen-Specific T Cells

Three samples of the tumor antigen-specific T cells derived from afrozen stock of tumor antigen-specific T cells prepared using Method 2mwere prepared and subjected to bulk and single-cell TCRα and TCRβamplification coupled to next-generation sequencing using the IPAIR™technology (iRepertoire, Inc.). The three samples are: (1) tumorantigen-specific T cells stimulated by an irrelevant peptide; (2) tumorantigen-specific T cells stimulated by RGS5-OLP5; and (3) INFγ⁺CD3⁺tumor antigen-specific T cells stimulated by RGS5-OLP5 enriched bybeads. As a control, a PBMC sample from the same patient was alsosubjected to the same next-generation sequencing analysis.

FIGS. 18A and 18B show the frequency of unique clonotypes of TCRα andTCRβ sequences in the various samples. Tumor antigen-specific T cellsstimulated by RGS5-OLP5 and INFy sorted tumor antigen-specific T cellsstimulated by RG5-OLP5 have much higher frequency of unique clonotypesof TCRα and TCRβ sequences than the control T cells, i.e., PBMCs andtumor antigen-specific T cells stimulated by an irrelevant peptide.

Notably, using the IPAIR™ Analyzer software, cognate TCRα and TCRβpairing was achieved for 69 out of 96 single-cell TCRα and TCRβsequencing samples (FIG. 19 ). A pair of RGS5-specific TCRα and TCRβgenes (clone 4) was identified from the 96-well plate shown in FIG. 19 .Three pairs of RGS5-specific TCRα and TCRβ genes (clones 4-6) from themost predominant clonotypes were identified and synthesized. See Table 1for detailed information of the exemplary TCR clones. Engineered T cellsexpressing each pair of RGS5-specific TCRα and TCRβ genes are prepared,and the TCRs are validated by assessing RGS5-specific immune response bythe engineered T cells.

Example 4: Validation of RGS5 and HPV18 E7-Specific TCRs

Tumor antigen-specific TCRs identified from Examples 2 and 3 werevalidated using assays shown in FIG. 23 . Five RGS5-specific TCRs andfive HPV18-E7-specific TCRs were validated.

Human (i.e., wildtype) TCR and murinized TCR constructs corresponding tothe were prepared (FIG. 24 ). The murinized TCR constructs have murineconstant domains (mCα and mCβ1). 3 TCR variants having an N-terminal19-amino acid leader sequence (SEQ ID NO: 101) were discovered and theirmurinized versions were prepared. FIGS. 25A-25B show the 26 TCRconstructs that were prepared and validated. Multiple TCRs recognizingthe same epitopes such as amino acids 16-30 of RGS5 and amino acids84-102 of HPV18-E7 were discovered. Validation assay results for the TCRconstructs are shown in FIGS. 26A-35G.

TCR Expression Assay

TCR expression was assessed using a FACS assay that detected stained TCRVβ chain. Briefly, TCR transferred T cells were collected and washed byadding 10 mL PBS (containing 2% Fetal Bovine serum), centrifuged at 350g for 5 minutes, and the supernatant was aspirated completely. Cellpellets were resuspended and adjusted to about 2 x10⁶ cells/mL by addingPBS (containing 2% Fetal Bovine serum). 100 µL/ sample cell suspensionwas collected and used for TCR Vβ chain surface staining and detected byFACS.

LCL Stimulation Assay

The LCL stimulation assay was carried out as follows. First, LCLs wereloaded with peptides: LCLs were collected in 15 mL tube, washed byadding 10 mL PBS, centrifuged at 350 g for 5 minutes, and thesupernatant was aspirated completely. Cell pellets were resuspended inculture medium (RPMI1640 containing 10% FBS), and adjusted to 1×10⁶cells/mL. Tumor antigen peptide was added to LCLs to a finalconcentration of 5 µg/mL, and incubated for 8-24 hours in incubator.TCR-T cells were then stimulated with peptide-loaded LCLs as follows:Peptide-loaded LCLs were collected and washed by adding 10 mL PBS,centrifuged at 350 g for 5 minutes, and the supernatant was aspiratedcompletely. The LCLs were resuspended and adjusted to 1×10⁶ cells/mL or1×10⁵ cells/mL by adding AIM-V medium (containing 10% Fetal Bovineserum). TCR-transduced T cells were collected, washed by adding 10 mLPBS, centrifuge at 350 g for 5 minutes, and the supernatant wasaspirated completely. TCR transduced T cells were then resuspended, andadjusted to 2×10⁶ cells/mL or 5×10⁵ cells/mL by adding AIM-V medium(containing 10% Fetal Bovine serum). 100 µL/ well of LCL (1×10⁶cells/mL) and 100 µL /well TCR-transduced T cells (2×10⁶ cells/mL) weremixed into a 96 wells plate. Brefeldin A (final concentration of 3µg/mL) was added to the wells, and incubated for 4 hours. After 4 hours,cells were collected and used for intracellular IFN-y and TNF-α stainingand detected by FACS. For ELISA detection of IFN-y, 100 µL/ well of LCL(1×10⁵ cells/mL) and 100 µL /well TCR-transduced T cells (5×10⁵cells/mL) were mixed into a 96 wells plate, and incubated for 24 hoursin an incubator. After 24 hours, 175 µL supernatant was collected perwell in the 96 well plate, and used in ELISA detection of IFN-γ using anIFN-y ELISA HRP Kit.

HLA Blocking Assay

HLA blocking assay was carried out as follows. Peptide loaded LCLs werecollected after LCLs stimulation, washed by adding 10 mL PBS,centrifuged at 350 g for 5 minutes, and the supernatant was aspiratedcompletely. Cell pellets were resuspended, adjusted to 1 × 10⁵ cells/mLby adding AIM-V medium (containing 10% Fetal Bovine serum). 100 µL/wellcell suspension was added to 96 wells plate, and HLA blocking antibodywas added (final concentration of 50 µg/mL) to appropriate wells, andincubated for 2 hours in an incubator. TCR transferred T cells werecollected, washed by adding 10 mL of PBS, centrifuged at 350 g for 5minutes, and the supernatant was aspirated completely. Cell pellets werecollected and adjusted to 5 × 10⁵ cells/mL by adding AIM-V medium(containing 10% Fetal Bovine serum). 100 µL/well TCR transduced cellsuspension was added to the corresponding well, mixed with the LCLs andincubated for 24 hours in an incubator. After 24 hours, 175 µLsupernatant was collected per well of the 96 well plate, and subjectedto ELISA detection of IFN-γ using an IFN-y ELISA HRP Kit.

HLA Restriction Assay

LCLs from different donors (Table 4) with various HLA-II genotypes wereloaded with peptides to stimulate TCR-T in the HLA restriction assay.The rest of steps were the same as LCL stimulation assay.

TABLE 4 Donors with different HLA genotypes. Donor 1 2 3 4 5 6 7 8 9 10MLA-DR DRA/DRB1*09:01 + + + - - - - + - -DRA/DRB4_(*)01:03 + + - + + - - + + + HLA-DPHLA-DPA1*02:02/DPB1*05:01 + + + - - + - - - +HLA-DPA1*01:03/DPB1*02:01 - - - - + - + + + -HLA-DPA1*01:03/DPB1*05:01 - - + + - - - - - -HLA1-DPA1*02:02/DPB1*02:01 - - - - - - + + - - HLA-DQDQAI*03:02/DQB1*03>03 + + + - - - - + - -

Human IFN-y ELISA

On Day 1, a high protein binding ELISA plate was coated with antibody1-D1K (IFN-y ELISA HRP Kit), diluted to 2 µg/mL in PBS, pH 7.4, byadding 50 µL/well, and incubated overnight at 4° C. On Day 2, the platewas washed twice with PBS (200 µL/well). The plate was blocked by adding200 µL/well culture medium, and incubated for 1 hour at room temperature(RT). Human IFN-y standard (IFN-y ELISA HRP Kit) was prepared in 2 mLPBS with 1% BSA to a concentration of 0.5 µg/mL, and left at RT for 15minutes and then the tube was vortexed. 50 µL/well of samples orstandards was diluted in culture medium and incubated for 2 hours at RT.The samples and standard probes were tested in duplicates. The plate waswashed five times with PBS containing 0.05% Tween 20. 50 µL/well ofantibody 7-B6-1-biotin (IFN-y ELISA HRP Kit) was added at 1 µg/mL inPBS, incubated for 1 hour at RT, and washed. 50 µL/well ofStreptavidin-HRP (IFN-y ELISA HRP Kit) diluted 1:1000 in PBS was added,incubated for 1 hour at RT, and washed. 100 µL/well of TMB substratesolution was added, incubated at RT at dark place for 15-30 minutes tillsolution in wells turn visible blue. 50 µL/well of stop solution wasadded to stop enzymatic reaction. The solution color changed from blueto yellow. The optical density was measured in an ELISA reader at 450nm.

Example 5: Two-Round Tumor Specific T Cell Amplification From a PatientTreated with MASCT

Patient SMZ was diagnosed with metastatic lung cancer, and received 5cycles of improved MASCT treatment (see, PCT/CN2018/081338 andPCT/CN2019/080535) with activated T cells prepared using DCs loaded witha pool of general tumor antigen peptides (hTERT, p53, Survivin,NY-ESO-1, CEA, CDCA1, VEGFR1, VEGFR2, RGS5, CCND1, MUC1, Her2, MAGEA1,MAGEA3, WT-1) and neoantigen peptides (SMX-1, SMX-2 and SMX-3). The toppanel of FIG. 36 shows antigen-specific T cell response by the patient’sPBMC sample after the 5^(th) cycle of improved MASCT in an ELISPOTassay. Four tumor antigens, hTERT, CCND1, MAGE-A1 and WT-1, inparticular, induced strong immune response. The patient was subsequentlytreated with an additional cycle (cycle 6) of improved MASCT. The bottompanel of FIG. 36 shows antigen-specific T cell response by the patient’sPBMC sample after the 6^(th) cycle of improved MASCT in an ELISPOTassay. In the ELISPOT assay, single peptides from each of the tumorantigen peptide sub-pools corresponding to antigens hTERT, CCND1,MAGE-A1, and WT-1, were used to detect antigen-specific immune responsesand to identify immune-dominant tumor antigen peptides. Peptides hTERT-1and hTERT-2 showed particularly strong immune response.

PBMC samples from Patient SMZ were obtained to prepare tumor specific Tcells using a two-round protocol.

Round 1

FIG. 37 provides an overview of the protocol for Round 1 oftumor-specific T cells preparation used in this example. Briefly, on Day1, peripheral blood mononuclear cells (PBMCs) from the patient wereobtained by density gradient centrifugation on Lymphoprep (NycomedPharma, Oslo, Norway). The adherent monocytes were continued to becultured in AIM-V medium with 1000 U/mL GM-CSF and 500 U/mL IL-4 todifferentiate into immature dendritic cells (DCs). The resultingimmature DCs were pulsed with a peptide pool comprising two tumorantigen peptides derived from hTERT (i.e., hTERT1 and hTERT2, 1µg/mL/peptide), and then cultured in a DC maturation medium todifferentiate into mature DCs. On Day 8, PBMCs were stimulated with thepeptide pool. On Day 9, an IFNy secretion assay-cell enrichment anddetection kit (Miltenyi Biotec) was used to isolate a population ofIFNγ⁺ T cells from the stimulated PBMCs. On Day 9, the IFNγ⁺ T cellswere co-cultured with antigen-loaded mature DCs in a medium containingIL-2 and an anti-PD-1 antibody. On Day 11, the co-culture was stimulatedwith PBMCs pulsed with the peptide pool or each individual peptide. OnDay 12, an IFNy secretion assay-cell enrichment and detection kit(Miltenyi Biotec) was used to isolate a population of IFNγ⁺ T cells.Meanwhile, the antigen-loaded mature DCs (“DC sti”) or PBMCs (“PBMCsti”) were prepared and co-cultured with the IFNγ⁺ T cells. On Day 14,an anti-CD3 antibody (OKT3) and IL-2 (at least about 2000 IU/mL) wereadded to the co-culture, which was continued to be cultured to Day 31 toobtain tumor antigen-specific T cells.

Cell proliferation was assessed using cell samples from Day 1 (PBMCs),Day 9 (before IFNy enrichment), Day 12 (after IFNy enrichment), and Days23, 29 and 30 (co-culture of IFNγ⁺ T cells with antigen-loaded DCs orPBMCs) by methods described in Example 2. As shown in FIG. 38A,protocols with antigen-loaded DCs or PBMCs yielded similar T cellproliferation results.

The IFNy production levels by tumor antigen-specific T cells in variousco-culture samples were determined by methods described in Example 2.FIG. 38B shows percentages of various T cell populations after theenrichment step. FIGS. 39A-39E compare the percentages of varioustumor-specific T cell populations in the cell samples on Days 23 and 30as determined by assessing IFNγ⁺ CD3⁺, IFNγ⁺ CD4⁺ and IFNγ⁺ TNFα⁺ cellsin response to stimulation by the tumor antigen peptide pool orindividual antigen peptides. Co-culture with antigen-loaded DCs yieldedthe highest percentages of tumor-specific T cells. As shown in FIG. 39F,98.6% of the IFNγ⁺ CD4⁺ cells in the sample on Day 30 are CCR7⁻ CD45RO⁻effector T cells.

Round 2

FIG. 40 provides an overview of the protocol for Round 2 oftumor-specific T cells preparation used in this example. Briefly, on Day1, tumor-specific T cells from Round 1 were cultured in a mediumcomprising a cytokine cocktail (IL-2, IL-7, and IL-15) and an anti-PD-1antibody. PBMCs or mature DCs loaded with the hTERT-2 peptide wereprepared. The tumor-specific T cells and the antigen-loaded DCs or PBMCswere co-cultured at a ratio between T cells and PBMCs of 1:3, 1:1 or 3:1or at a ratio between T cells and DCs of 3:1 and 1:1. On Day 3, ananti-CD3 antibody (OKT3) and IL-2 were added to the co-culture, whichwas continued to be cultured to Day 8 to obtain tumor antigen-specific Tcells.

Cell proliferation was assessed using cell samples from Day 1 (round 1tumor-specific T cells), and Day 8 (co-culture of IFNγ⁺ T cells withantigen-loaded DCs or PBMCs) by methods described in Example 2. The IFNyproduction levels by tumor antigen-specific T cells in variousco-culture samples were determined by methods described in Example 2. Asshown in FIG. 41A, co-culture with antigen-loaded DCs yielded thehighest number of T cells and highest percentage of tumor-specific Tcells on Day 8.

FIGS. 41B-41C compare the percentages of various tumor-specific T cellpopulations in the cell samples on Day 8 as determined by assessingIFNγ⁺ CD3⁺ and IFNγ⁺ TNFa⁺ cells in response to stimulation by thehTERT2 antigen peptide. Co-culture with antigen-loaded DCs with a ratiobetween DCs to T cells of 3:1 yielded the highest percentages oftumor-specific T cells.

FIG. 42 shows the number of T cells and tumor-specific T cells on Day 1and Day 30 of Round 1, and on Day 38, i.e., Day 8 of Round 2. Thetwo-round protocols are effective in amplifying tumor-specific T cells.Stimulation by antigen-loaded DCs in Round 2 yielded the higher numberof T cells and higher percentage tumor-specific T cells than stimulationby antigen-loaded PBMCs. The tumor-specific T cells obtained at the endof Round 1 are effector T cells.

Table 5, below, provides the CDR sequences of exemplary TCRs.

TABLE 5 CDR sequences of Exemplary TCRs TCR ID TCR chain CDR1 SEQ ID NOCDR2 SEQ ID NO CDR3 SEQ ID NO P09E06 Alpha TISGNE 254 GLKNN 255CIVRAWYNNNDMRF 28 Beta SGHAT 256 QFQNNGV 257 CASRDTEAFF 31 09D01 AlphaNSASQS 258 VYSSGN 259 CVVNMRDSSYKLIF 34 Beta DFQATT 260 SNEGSKA 261CSAHERITDTQYF 37 09H05 Alpha NSASQS 262 VYSSGN 263 CVVNMKDSSYKLIF 40Beta DFQATT 264 SNEGSKA 265 CSALEGTSGKETQYF 43 09E01 Alpha TTLSN 266LVKSGEV 267 CAGPGNQFYF 46 Beta MNHEY 268 SVGAGI 269 CASSSWDRDQPQHF 4909B03 Alpha YSGSPE 270 HISR 271 CALSALPYNQGGKLIF 52 Beta GTSNPN 272SVGIG 273 CAWARSRELFF 55 P09B08 Alpha NSAFQY 274 TYSSGN 275CAFYAGNNRKLIW 58 10F04 Alpha NSAFQY 278 TYSSGN 279 CAMSPRSGYALNF 70 BetaLNHDA 280 SQIVND 281 CASSMDAALGEKLFF 73 09B12 Alpha ATGYPS 282 ATKADDK283 CALRSGGSNYKLTF 76 Beta SGDLS 284 YYNGE 285 CASSVEWGTYEQYF 79 33A02Alpha YGATPY 286 YFSGDTLV 287 CAVYQGAQKLVF 87 Beta DFQATT 288 SNEGSKA289 CSAPWLAGLYNEQFF 90 33D05 Alpha SSYSPS 290 YTSAATLV 291CVVSAIGYSSASKIIF 93 Beta SEHNR 292 FQNEAQ 293 CASSLVAGGPAETQYF 96

What is claimed is:
 1. A tumor-specific TCR comprising an antigen binding domain that binds to an epitope of HPV18-E7 comprising the amino acid sequence of SEQ ID NO: 85, wherein the TCR is selected from the group consisting of the following TCRs: (a) a TCRα chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 274, a CDR2 comprising the amino acid sequence of SEQ ID NO: 275, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 58; and a TCRβ chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 276, a CDR2 comprising the amino acid sequence of SEQ ID NO: 277, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 61; (b) a TCRα chain comprising a CDR1 comprising amino acid residues 27 to 38 of SEQ ID NO: 65, a CDR2 comprising amino acid residues 56 to 65 of SEQ ID NO: 65, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 64, and a TCRβ chain comprising a CDR1 comprising amino acid residues 27 to 38 of SEQ ID NO: 68, a CDR2 comprising amino acid residues 56 to 65 of SEQ ID NO: 68, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 67; (c) a TCRα chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 278, a CDR2 comprising the amino acid sequence of SEQ ID NO: 279, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a TCRβ chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 280, a CDR2 comprising the amino acid sequence of SEQ ID NO: 281, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 73; (d) a TCRα chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 282, a CDR2 comprising the amino acid sequence of SEQ ID NO: 283, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 76; and a TCRβ chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 284, a CDR2 comprising the amino acid sequence of SEQ ID NO: 285, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 79; (e) a TCRα chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 286, a CDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87; and a TCRβ chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 288, a CDR2 comprising the amino acid sequence of SEQ ID NO: 289, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 90; and (f) a TCRα chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 290, a CDR2 comprising the amino acid sequence of SEQ ID NO: 291, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 93; and a TCRβ chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 292, a CDR2 comprising the amino acid sequence of SEQ ID NO: 293, and a CDR3 comprising the amino acid sequence of SEQ ID NO:
 96. 2. The tumor-specific TCR of claim 1, wherein the TCR comprises the TCRα chain comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 278, the CDR2 comprising the amino acid sequence of SEQ ID NO: 279, and the CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the TCRβ chain comprising the CDR1 comprising the amino acid sequence of SEQ ID NO: 280, the CDR2 comprising the amino acid sequence of SEQ ID NO: 281, and the CDR3 comprising the amino acid sequence of SEQ ID NO:
 73. 3. The tumor-specific TCR of claim 1, wherein the TCR comprises the TCRα chain comprising a CDR1 comprising amino acid residues 27 to 38 of SEQ ID NO: 65, a CDR2 comprising amino acid residues 56 to 65 of SEQ ID NO: 65, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 64, and a TCRβ chain comprising a CDR1 comprising amino acid residues 27 to 38 of SEQ ID NO: 68, a CDR2 comprising amino acid residues 56 to 65 of SEQ ID NO: 68, and a CDR3 comprising the amino acid sequence of SEQ ID NO:
 67. 4. The tumor-specific TCR of claim 1, wherein the TCR comprises the TCRα chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 278, a CDR2 comprising the amino acid sequence of SEQ ID NO: 279, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a TCRβ chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 280, a CDR2 comprising the amino acid sequence of SEQ ID NO: 281, and a CDR3 comprising the amino acid sequence of SEQ ID NO:
 73. 5. The tumor-specific TCR of claim 1, wherein the TCR comprises the TCRα chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 282, a CDR2 comprising the amino acid sequence of SEQ ID NO: 283, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 76; and a TCRβ chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 284, a CDR2 comprising the amino acid sequence of SEQ ID NO: 285, and a CDR3 comprising the amino acid sequence of SEQ ID NO:
 79. 6. The tumor-specific TCR of claim 1, wherein the TCR comprises the TCRα chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 286, a CDR2 comprising the amino acid sequence of SEQ ID NO: 287, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87; and a TCRβ chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 288, a CDR2 comprising the amino acid sequence of SEQ ID NO: 289, and a CDR3 comprising the amino acid sequence of SEQ ID NO:
 90. 7. The tumor-specific TCR of claim 1, wherein the TCR comprises the TCRα chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 290, a CDR2 comprising the amino acid sequence of SEQ ID NO: 291, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 93; and a TCRβ chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 292, a CDR2 comprising the amino acid sequence of SEQ ID NO: 293, and a CDR3 comprising the amino acid sequence of SEQ ID NO:
 96. 8. The tumor-specific TCR of claim 1, comprising: (a) a TCRα chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 59, and a TCRβ chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 62; (b) a TCRα chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 65, and a TCRβ chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 68; (c) a TCRα chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 71, and a TCRβ chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 74; (d) a TCRα chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 77, and a TCRβ chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 80; (e) a TCRα chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 88, and a TCRβ chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 91; or (f) a TCRα chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO: 94, and a TCRβ chain comprising an amino acid sequence having at least about 80% identity to the amino acid sequence of SEQ ID NO:
 97. 9. The tumor-specific TCR of claim 1, wherein the TCRα chain comprises the amino acid sequence of SEQ ID NO: 59, and the TCRβ chain comprises the amino acid sequence of SEQ ID NO:
 62. 10. The tumor-specific TCR of claim 1, wherein the TCRα chain comprises the amino acid sequence of SEQ ID NO: 65, and the TCRβ chain comprises the amino acid sequence of SEQ ID NO:
 68. 11. The tumor-specific TCR of claim 1, wherein the TCRα chain comprises the amino acid sequence of SEQ ID NO: 71, and the TCRβ chain comprises the amino acid sequence of SEQ ID NO:
 74. 12. The tumor-specific TCR of claim 1, wherein the TCRα chain comprises the amino acid sequence of SEQ ID NO: 77, and the TCRβ chain comprises the amino acid sequence of SEQ ID NO:
 80. 13. The tumor-specific TCR of claim 1, wherein the TCRα chain comprises the amino acid sequence of SEQ ID NO: 88, and the TCRβ chain comprises the amino acid sequence of SEQ ID NO:
 91. 14. The tumor-specific TCR of claim 1, wherein the TCRα chain comprises the amino acid sequence of SEQ ID NO: 94, and the TCRβ chain comprises the amino acid sequence of SEQ ID NO:
 97. 15. The tumor-specific TCR of claim 1, comprising: (a) a TCRα chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 226, and the TCRβ chain comprises a variable region comprising the amino acid sequence of SEQ ID NO: 224; (b) a TCRα chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 234, and the TCRβ chain comprises a variable region comprising the amino acid sequence of SEQ ID NO: 232; (c) a TCRα chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 218, and the TCRβ chain comprises a variable region comprising the amino acid sequence of SEQ ID NO: 216; (d) a TCRα chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 246, and the TCRβ chain comprises a variable region comprising the amino acid sequence of SEQ ID NO: 244; or (e) a TCRα chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 230, and the TCRβ chain comprises a variable region comprising the amino acid sequence of SEQ ID NO:
 228. 16. An engineered immune cell comprising the tumor-specific TCR of claim
 1. 17. The engineered immune cell of claim 16, wherein the immune cell is a T cell.
 18. A pharmaceutical composition comprising the engineered immune cell of claim 17, and a pharmaceutically acceptable carrier. 